METHOD FOR IDENTIFYING THE PRESENCE AND / OR CONCENTRATION AND / OR AMOUNT OF PROTEINS OR PROTEOMS

Description

FIELD OF THE INVENTION

[0001] The present invention relates to methods of identifying the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest within a sample.BACKGROUND OF THE INVENTION

[0002] Proteins are biological polymers that are comprised of sequences of amino acids. Proteomics is the large-scale study of proteins. It allows the identification of and quantification of proteins. Within the field of proteomics, there are multiple established methods to identify the presence or absence of a protein within a sample. Identification of the presence or absence of a subproteome or a proteome within a sample is challenging as this involves sequential identification of all of its proteins. Some proteomic methods allow the quantification of the concentration or amount of a protein within a sample.

[0003] The most common method for identifying the presence of proteins in a sample is mass spectrometry. Mass spectrometry measures the mass-to-charge ratio of ions present in a sample. The mass spectrum of a sample is a plot of the ion signal as a function of the mass-to-charge ratio. The spectra are used to determine the isotopic signature of a sample and the masses of particles, which are used to provide the chemical identity or structure of chemical compounds. However, mass spectrometry is labour intensive and is not inherently quantitative because different peptides are ionized and detected with different efficiencies. To combat this, approaches such as isotope-coded affinity tags (ICAT) are used, but this only permits a fraction of proteins identified to be quantified. Most quantitative mass spectrometry approaches permit determination of only relative changes in protein concentration or amount across samples, rather than absolute quantification of samples. Mass spectrometry proteomics is also limited in coverage, particularly for higher organisms. 'Top down' mass spectrometry proteomics which analyses whole proteins only permits protein identification for 10% of the proteins studied, and 'bottom up' mass spectrometry proteomics which analyses proteins which have been digested into fragments permits protein identification for 8-25% of the proteins studied. Due to the complexity of the mass spectra obtained, mixtures and complex samples must be separated into their components, for example by two-dimensional gel electrophoresis or high-performance liquid chromatography (HPLC), before they can be sequentially analysed with mass spectrometry.

[0004] An alternative approach to identify the presence of proteins is to use protein microarrays. Protein microarrays immobilize an array of proteins, or an array of probes, onto a support surface and are particularly suitable for multiplexed detection. Tagged probes or tagged proteins are added to the array and the binding interaction between the protein and the probe is detected. However, protein microarrays are labor intensive and suffer from a lack of reproducibility and accuracy. Detection requires a binding event near a surface and therefore, the binding event and thus the accuracy of detection can be affected by the surface. Furthermore, only the proteins which already have a corresponding probe, such as a specific antibody, can be identified by this method.

[0005] Several methods have aimed to identify a protein via physical parameters characteristic of a protein, for example Zhang et al., "Top-down proteomics on a microfluidic platform" (2019), eprint 1910.11861 arXiv physics.bio-ph. In this microfluidic method, a protein's hydrodynamic radius (R H ) which is its size in solution is used with ratios of fluorescence signals from Trp / Lys and Tyr / Lys residues within proteins for protein identification. The lysine (Lys) residues are fluorescently labelled and the tryptophan (Trp) and tyrosine (Tyr) residues are unlabelled. Seven known proteins are measured four times, and a protein is identified when the values obtained the fourth time the protein is measured match the values obtained the other three times the protein was measured. While it is shown that the values measured are characteristic of a known protein under a set of experimental conditions in that the measured proteins are distinguishable from each other based on these values, none of the values can be predicted for a protein of interest. R H cannot be predicted for an amino acid sequence, which has unknown and often partial intrinsic disorder. Those skilled in the art appreciate that intrinsic fluorescence from tryptophan and tyrosine residues depends in a complex manner on the local physical environment surrounding the tryptophan and tyrosine residues within a protein structure which is currently unpredictable from an amino acid sequence. Therefore, R H , Trp and Tyr signals would all change with the solution conditions, for example different readings for the same protein would be obtained if the protein is placed in a different buffer or if it interacts with another biomolecule. The method does not allow protein quantification because none of the values used for protein identification provides information about protein amount or concentration. Due to the unpredictable nature of the results obtained, it is not possible to analyse a mixture of proteins or a proteome using this method.

[0006] Alternatively, the state-of-the-art includes newly developed protein sequencing methods such as Swaminathan, J et al. Nat Biotechnology 36, 1076-1082 (2018). Sparse fluorosequencing performs classical Edman degradation sequencing on single peptide fragment molecules that have been fluorescently labelled on specific amino acids prior to their immobilization onto a surface and observes the pattern of fluorescence disappearance from the surface as the fluorescently labelled amino acids are sequentially cleaved from the peptide N-terminus. The pattern of fluorescence decreases reveals the positions of the labelled amino acids within the peptide being read and provides a sparse peptide sequence. These sparse peptide sequences can be predicted for a protein of interest based on the information-rich constraints of protease cleavage specificity, surface attachment chemistry, labelling chemistry, and the positions of the labelled amino acids within the predicted peptide fragments for the protein of interest. Practically, this labor and data intensive method is prone to error from a variety of sources and correct reads are observed approximately 40% of the time for a single purified peptide. Quantification was not evaluated for this method. The method relies on coupling to chromatographic separation methods like HPLC and / or mass spectrometry to first verify that all amino acids, such as all lysine and all cysteine amino acids, are quantitatively fluorescently labelled within each peptide fragment prior to carrying out the sequencing. Although peptide fragments within a two-component mixture were identified, this requires that the peptide fragments be spatially separated from one another via surface attachment at different positions on the surface, so that distinct fluorescence disappearance traces can be observed for each peptide. Like traditional Edman sequencing, this method is slow with one Edman cycle requiring 1 hour, not suitable for the analysis of N-terminally modified peptides, and not suitable for reading peptide fragments greater than 30 amino acids in length. It is admitted that due to relying on Edman sequencing, this method is more suitable for the identification of short peptides rather than long protein molecules. The average length of a protein molecule within the human proteome is 558 amino acids. It is not possible to analyze a mixture of proteins or a proteome using this method.

[0007] There is a recognized need for the development of simple and general alternatives to mass-spectrometry based protein identification that permit the identification of whole proteins as proteins of interest. There is a recognized need for an efficient method of characterizing complex mixtures of proteins, for example mixtures of proteins that are disease-associated. There is a great need for a rapid and general method of diagnosing any infection. Preferably these methods would enable protein quantification. Therefore, there is a need for a more efficient, cost-effective and general method for identifying the presence and / or concentration and / or amount of proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest within a sample.SUMMARY OF INVENTION

[0008] The invention is as defined in the appended claims. The invention is based on the discovery that labelling and measuring three or more amino acid types in a sample can identify the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest in a sample. This is based on the measured label, amino acid concentration, or number of amino acids of each labelled amino acid type in the sample.

[0009] It has been discovered that each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome has a unique signature based on the label values, amino acid concentrations, or number of amino acids of three or more amino acid types for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome at each concentration.

[0010] The signature of the label values or amino acid concentrations of each of three or more amino acid types for a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome is unique for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome at each concentration. The signature of the number of amino acids of each of three or more amino acid types for a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome is also unique for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome.

[0011] Therefore, the signature of the sample can be compared to the signature of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest to identify the presence and / or concentration and / or amount of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest in the sample.

[0012] The signature of the known label values or amino acid concentrations of three or more amino acid types in a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is a function of the concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest, and is unique for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest at each concentration. Therefore, the values of the measured labels or amino acid concentrations of three or more amino acid types in the sample can be compared to the known label values or amino acid concentrations of the same three or more amino acid types that have been labelled in the sample for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest to provide a positive identification of the presence and / or concentration and / or amount of that protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest in the sample. The signature of the number of amino acids of three or more amino acid types in a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is unique for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. Therefore, the number of amino acids of each of three or more amino acid types in the sample can be compared to the number of amino acids of the same three or more amino acid types that have been labelled in the sample for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest to provide a positive identification of the presence in the sample.

[0013] This comparison is visualized using an n-dimensional space, where the number of dimensions is equal to the number of n amino acid types labelled and measured in the methods of the invention. For example, three labelled amino acid types are visualized in a 3-dimensional space. This dimensional space increases as each additional amino acid type is labelled and measured in the sample. The amino acid concentrations or values of the label of the three or more amino acid types take on a line in n-dimensional space. The number of amino acids of each the three or more amino acid types take on a point in n-dimensional space. There are n dimensions for n amino acid types labelled in the sample.

[0014] It has been discovered that the label, amino acid concentration, or number of amino acids of only three or more amino acid types need to be measured in order to identify the presence and / or concentration and / or amount of a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome, of interest in the sample. Labelling and measuring three or more amino acid types is essential to the methods of the invention because when three or more amino acid types are labelled and measured, this provides the unique signature for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. At least two amino acid types are required to be labelled and measured because if only one amino acid type were labelled and measured, all proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest would have the same reference line. When the sample point is compared to p lines for p proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest, each a function of concentration, the presence and / or concentration and / or amount of a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest within the sample is simultaneously determined. In this solution phase method, the amount of a protein contained within the sample is simply determined by multiplying the concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome identified within the sample by the volume of solution within the sample. It is not necessary or efficient to measure the label, amino acid concentration, or number of amino acids for every amino acid type in the sample.

[0015] Proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, and proteomes of interest all have unique signatures of the known values of the label, amino acid concentrations, or number of amino acids of three or more amino acid types. It is not necessary to know or suspect what category of molecules the sample contains (i.e. a protein, peptide, oligopeptide, polypeptide, protein complex, mixture, subproteome, or proteome) to determine the presence and / or concentration and / or amount of a member of that category of interest within the sample. For example, the three or more amino acid types labelled in the sample may include tryptophan (W) and lysine (K), the measured label of tryptophan (W) is used to determine the concentration of tryptophan (W) in the sample, and the measured label of lysine (K) is used to determine the concentration of lysine (K) in the sample. The sample contains 10.9 µM W and 27.9 µM K. The sample is identified against the protein of interest hen egg white lysozyme and the proteome of interest HIV. Hen egg white lysozyme has 6 W and 6 K amino acids per protein sequence and HIV has 10.9 W amino acids and 27.9 K amino acids per protein sequence. The absence of hen egg white lysozyme in the sample is identified because there is no protein concentration of hen egg white lysozyme which would result in measuring the signature of the sample. However, the signature of the sample (10.9 µM W and 27.9 µM K) is the same as the signature of HIV (10.9 W and 27.9 K) at 1 µM protein concentration, and so the presence of 1 µM HIV in the sample is identified.

[0016] It is the label, amino acid concentration, or number of each labelled amino acid type in the sample compared to the known label values, amino acid concentrations, or number of amino acids, respectively, of the same amino acid types in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest that is important, and not the order of the amino acids in the sample compared to the order of the amino acids in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. Other state-of-the-art methods for peptide and protein identification require determination of the order of the amino acids within peptide or protein sequences of the sample.

[0017] Three or more amino acid types in the sample are labelled. An amino acid type is defined by the R-group, i.e. side chain. The R-group is specific to each amino acid type. The R-group of one amino acid type is distinguishable from the R-group of every other amino acid type. For example, R-group for tryptophan (W) is an indole group. Every W amino acid has an indole group. Therefore, the W amino acid type is defined by the indole R-group. In another example, the R-group for lysine (K) is a ε-primary amino group. Every K amino acid has this ε-primary amino group. Therefore, the K amino acid type is defined by the ε-primary amino R-group. In another example, the R-group for tyrosine (Y) is a phenol group. Every Y amino acid has a phenol group. Therefore, the Y amino acid type is defined by the phenol R-group. The R-group of the amino acid type W is distinguishable to the R-group of the amino acid type K and the R-group of the amino acid type Y. Hence, the amino acid type W is distinguishable to the amino acid type K and the amino acid type Y because of the different R-groups between these amino acid types. All the amino acid types are distinguishable from each other by their specific R-group. In some embodiments, an amino acid type is labelled independently to the other amino acid types. In some embodiments, it is the R-group of each amino acid of an amino acid type that is labelled. In some embodiments, each R-group (i.e. each amino acid type) has a unique label and so each R-group (i.e. each amino acid type) is labelled independently to the other R-groups (i.e. other amino acid types). In some embodiments, two or more R-groups (i.e. two or more amino acid types) are labelled with the same label, but each labelled R-group (i.e. each labelled amino acid type) is detected differently to another labelled R-group (i.e. another labelled amino acid type). In some embodiments, each label is targeted to an amino acid type. In some embodiments, each label is specific for an amino acid type.

[0018] In some embodiments, the three or more amino acid types are selected from alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), pyrrolysine (S), selenocysteine (O), threonine (T), tryptophan (W), tyrosine (Y) and valine (V) or synthetic amino acids. In some embodiments, an amino acid type comprises modified amino acids and / or unmodified amino acids. In some embodiments, an amino acid type comprises modified amino acids. In some embodiments, an amino acid type comprises unmodified amino acids. In some embodiments, an amino acid type comprises both modified and unmodified amino acids. In some embodiments, when both the modified and unmodified amino acids of an amino acid type are labelled, the modified amino acids are first converted into unmodified amino acids.

[0019] In some embodiments, proteins within the sample are fluorogenically labelled with molecules whose fluorescence "turns on" exclusively after reaction with the amino acid type of interest. Therefore, separation of labelled amino acids from unreacted dye is not required, because the unreacted dye is not fluorescent and does not provide a signal. In other state of the art methods for peptide or protein identification, separation of labelled amino acids from unreacted dye is required before peptide or protein identification can take place.

[0020] The label of each labelled amino acid type in the sample is measured. For example, if the three or more amino acid types labelled in the sample include tryptophan (W) and lysine (K), then the label of tryptophan (W) is measured, and the label of lysine (K) is measured.

[0021] In some embodiments, the measured label of each amino acid type is used to calculate the concentration of that labelled amino acid type and / or the number of amino acids of that labelled amino acid type in the sample. The measured label of each amino acid type can be linearly related to each of the concentration of the amino acid type, the number of amino acids of the amino acid type, and the concentration of the sample. For example, if the three or more amino acid types labelled in the sample include tryptophan (W) and lysine (K), then the label of tryptophan (W) is measured, and the label of lysine (K) is measured. The measured label of tryptophan (W) is used to calculate the amino acid concentration of tryptophan (W) and / or the number of tryptophan (W) amino acids in the sample and / or the concentration of the sample. The measured label of tryptophan is linearly related to each of the concentration of tryptophan amino acids, the number of tryptophan amino acids, and the concentration of the sample. The measured label of lysine (K) is used to calculate the amino acid concentration of lysine (K), and / or, the number of lysine (K) amino acids, and / or the concentration of the sample. The measured label of lysine is linearly related to each of the concentration of lysine, the number of lysine amino acids, and the protein concentration of the sample.

[0022] In some embodiments, a calibration curve or standard is used to convert the values of the measured label (e.g. signals) into amino acid concentrations for each of three or more amino acid types labelled in the sample. A calibration curve or standard shows how the response of an instrument changes with the known concentration of an analyte. A standard or calibration curve provides the values of the label for one or more known amino acid concentrations of each amino acid type. This conversion can be applied to the sample or the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. For example, the calibration curve reveals that for the amino acid type tryptophan (W), to determine the known value of the label for a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest at an amino acid concentration of 10 µM W, this amino acid concentration is multiplied by 100 AU uM because that is the slope of the calibration curve. The calculation indicated by the calibration curve or standard is called a calibration function or a calibration factor. A calibration factor is used if the values are multiplied or divided by a scalar, and a calibration function is used if additional steps are performed. For example, 100 AU uM is a calibration factor. There is no requirement to calculate the calibration curve or standard each time a sample is measured, instead these curves or standards can be supplied to the user who only needs to measure the label (e.g. signal) of three or more labelled amino acid types in his sample and can be provided with the calibration function or factor for each amino acid type. In this embodiment, the positive identification of the presence and / or concentration and / or amount of the sample is based on the concentration of amino acids of each labelled amino acid type of the sample. The measured label of each labelled amino acid type in the sample can be linearly related to the concentration of that amino acid type in the sample, the number of amino acids per protein of that amino acid type in the sample, and / or the protein concentration of the sample.

[0023] In some embodiments, the number of amino acids of each labelled amino acid type in the sample is calculated by dividing the amino acid concentration of each labelled amino acid type by the molar protein concentration of the sample. Therefore, it is necessary to know the molar protein concentration of the sample in order to use the value of the number of amino acids in the sample. In this embodiment, the positive identification of the presence of a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest within the sample can be based on the number of amino acids of each labelled amino acid type in the sample.

[0024] If the amino acid concentrations or known label values of n amino acids for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest are plotted as a function of its concentration, this provides a line in n-dimensional space, from which the concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest in the sample can be determined using the equation of the line. In some embodiments, the line originates at the origin. In alternative embodiments, the line comprises the amino acid concentrations or known label values corresponding to concentrations within a known concentration range. The amino acid concentrations or measured label for the labelled amino acid types in the sample take on a point in n-dimensional space. The point of the sample can be compared to the line in the n-dimensional space to identify the presence and / or concentration and / or amount of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest in the sample.

[0025] For example, if there are 4 proteins of interest; protein-A, protein-B, protein-C, and protein-D represented in 2-dimensional space, where dimension one and two are the label values for cysteine (C) and tryptophan (W) respectively. The cysteine (C) and tryptophan (W) amino acid types are labelled in the sample and measured. Figure 1 plots the measured label values of the cysteine (C) and tryptophan (W) amino acid types labelled in the sample as a point in 2-dimensional space, against the known label values of cysteine (C) and tryptophan (W) represented as a line in 2-dimensional space for each of the four proteins of interest respectively. The known label values of the cysteine (C) and tryptophan (W) amino acid types are plotted as a function of protein concentration for proteins of interest; protein-A, protein-B, protein-C and protein-D. The known label values take on a distinct line in 2-dimensional space for each of the four proteins of interest.

[0026] In some embodiments, this line is a reference line. In Figure 1, each point on the reference line of each of the four proteins of interest corresponds to a concentration of the respective protein of interest. As the protein concentration of a protein of interest increases, the known label values of each amino acid type provided by its reference line move further from its origin. The points corresponding to a concentration of 1 µM of each protein of interest are shown with shaded circles. The value of the label of each of the cysteine (C) and tryptophan (W) amino acid types in the sample is measured, and this point is shown with an open square. In some embodiments, the shortest distance between the sample point and each reference line is calculated.

[0027] In some embodiments, the sample point lies on the reference line for a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. The presence of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest within the sample is identified, and the concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is the concentration for which the measured value of the label or amino acid concentration of each of the three or more amino acid types labelled in the sample is equivalent to the known value of the label or amino acid concentration of each of the same three or more amino acid types as were labelled in the sample.

[0028] In other embodiments, the sample point is not on the reference line, and the distance between the sample point and the reference line is calculated. In some embodiments, this distance is the length of a vector or line segment to the reference line, connecting the sample point and the reference line. The sample point is closest to a single point on the reference line for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest, corresponding to the amino acid concentrations or known values of the label of n amino acid types for a single concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest.

[0029] In some embodiments, the presence of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is identified in the sample if the distance between the sample point and this closest point on the reference line for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is less than or equal to an error margin. In some embodiments, the error margin is a distance threshold. If the presence of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is identified within the sample, then it is present at the protein concentration of the point on the reference line to which the sample point was closest.

[0030] In Figure 1, the shortest distance between the sample point and the four reference lines corresponding to the four proteins of interest was the distance between the sample point and the reference line of protein-B. The presence of protein of interest protein-B in the sample is identified. Each point on the reference line for protein of interest protein-B shows the value of the label of the cysteine (C) and tryptophan (W) amino acid types for a distinct protein concentration of protein of interest protein-B. The sample is identified as the protein concentration of the point on the reference line of protein-B which provided the smallest distance. Here, the protein concentration of the sample is 0.5 µM. Therefore, a positive identification of protein of interest protein-B in the sample can be made, and the concentration of protein of interest protein-B at 0.5 µM within the sample is simultaneously determined.

[0031] In some embodiments, if molar protein concentration of the sample is known and so the value of the number of amino acids of three or more amino acid types in the sample is available, then the number of amino acids of the same corresponding three amino acid types are plotted in n-dimensional space, providing a point for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest.

[0032] There is only one point for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. Therefore, the point of the sample can be compared to the point for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest and the presence of a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is identified in the sample if the point of the sample is the same as the point for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. In some embodiments, the distance between the sample point and the point for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest can be calculated, and the presence of a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is identified in the sample if the distance between the sample point and the point for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is less than or equal to an error margin.

[0033] In some embodiments, if the measured label and / or concentration and / or number of amino acids of each labelled amino acid type in the sample is equivalent to, or within an error margin to the known label values and / or concentrations and / or number of amino acids of the same amino acid types as were labelled in the sample in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest, then a positive identification of the presence and / or concentration and / or amount of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest in the sample can be made. For example, if the amino acid concentration of tryptophan (W) amino acids and the amino acid concentration of lysine (K) amino acids in the sample is equivalent to, or within an error margin to the amino acid concentration of tryptophan (W) amino acids and the amino acid concentration of lysine (K) amino acids for a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest, then a positive identification of the presence and / or concentration and / or amount of that protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest in the sample can be made.

[0034] In some embodiments, the minimum distance between the measured value of the label, amino acid concentration, or number of amino acids of three or more amino acid types labelled in the sample and the known values of the label, amino acid concentrations, or number of amino acids of three or more amino acid types provided for a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is calculated, and this distance is compared to the error margin.

[0035] In some embodiments, the known label values, amino acid concentrations and / or number of amino acids of three or more amino acid types provided for each of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is a reference. In some embodiments, the reference is obtained from a database. Alternatively, the reference can be calculated.

[0036] The unit of each labelled amino acid type (i.e measured label, amino acid concentration and / or number of amino acids) in the sample must be compared to the same unit of the same amino acid types (i.e known label values, amino acid concentrations and / or number of amino acids) in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest (e.g. reference). For example, if the number of amino acids of W and Y are determined in the sample, then this must be compared to the number of amino acids of W and Y in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest (e.g. reference) so that the unit (number of amino acids) of the sample is compared to the same unit (number of amino acids) of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. If the amino acid concentration of W and Y are determined in the sample, then this must be compared to the amino acid concentration of W and Y in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest (e.g. reference) so that the unit (amino acid concentration) of the sample is compared to the same unit (amino acid concentration) of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest.. If the measured label of W and Y in the sample is not used to determine the amino acid concentration or the number of amino acids of W and Y in the sample, then the measured label of W and Y in the sample must be compared to the known label value of W and Y for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest ( e.g. reference) so that the unit (measuring the label) of the sample is compared to the same unit (the known label value) of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. For example, the measured fluorescence intensity of W and Y in the sample is compared to the known fluorescence intensity of W and Y in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest (e.g. reference).

[0037] In some embodiments, if the units measured for the sample (i.e measured label, amino acid concentration and / or number of amino acids) are different to the units (i.e known label value, amino acid concentration and / or number of amino acids) of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest, then the unit of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is converted into the same unit that has been measured for the sample. In some embodiments, the number of amino acids of a particular amino acid type of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is multiplied by the concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome to provide the amino acid concentration of each amino acid type in the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest. For example, if the amino acid concentration of W and Y has been measured in the sample, then the number of W and Y amino acids in one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is converted into the corresponding amino acid concentration of W and Y in each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. This allows the unit of the sample to be compared to the same unit of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest, i.e. the measured amino acid concentration of W and Y in the sample to be compared to the amino acid concentration of W and Y in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest.

[0038] In some embodiments, the known label value, amino acid concentration and / or number of amino acids of the corresponding amino acid types in the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is calculated from the amino acid sequence or sequences and / or any experimental information about post-translation modifications of each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. In some embodiments, the amino acid sequence and / or any experimental information about post-translation modifications of each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is used to calculate the number of amino acids of each amino acid type that was labelled in the sample in the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest. For example, if the three or more amino acid types labelled in the sample include tryptophan (W) and lysine (K), then the number of tryptophan (W) amino acids and the number of lysine (K) amino acids in a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is calculated from the protein sequence or protein sequences of that protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. For example, if the three or more amino acid types labelled in the sample include tryptophan (W) and lysine (K) and the protein of interest in the sample is bovine serum albumin, then the number of tryptophan (W) and lysine (K) amino acids in the amino acid sequence of bovine serum albumin is calculated from the amino acid sequence of bovine serum albumin as 2W and 59K. As another example, if it is known via the methods disclosed herein that a protein of interest has 3 post-translational modifications on lysine (K) amino acids that make these lysine amino acids unreactive to the label, then -3 is added to the number of lysine amino acids of this protein of interest.

[0039] In some embodiments, the amino acid sequence or sequences of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is known (e.g. obtained from a database). In some embodiments, the amino acid sequence of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is determined using standard techniques of the art (e.g. Edman degradation or mass spectrometry).

[0040] In some embodiments, the number of amino acids of three or more labelled amino acid types of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is determined using the methods disclosed herein, i.e. labelling three or more amino acid types, measuring the label and using the measured label to determine the number of amino acids of each amino acid type in the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest, or the concentration of amino acids of each amino acid type in a sample containing each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. In this way, the presence and / or concentration and / or amount of proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest whose amino acid sequences are not known or not fully known can be determined.

[0041] In some embodiments, it is the number of each of the three or more amino acid types in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest, and not the order of each of the three or more amino acid types in the protein sequence or the relative composition of each of three or more amino acid types in the protein sequence, that is used to calculate the corresponding amino acid concentration and / or known label value of these amino acid types at one or more concentrations of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest.

[0042] It has been discovered that the unique signature of the known values of the labels or amino acid concentrations for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest can be provided with a vector function, or a set of parametric equations, depending on the common parameter of the concentration of each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. In some embodiments, this vector function or set of parametric equations describes and is used to calculate the reference line disclosed herein, such that the reference line can be quantitatively compared to a sample point in order to identify the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest within a sample. A set of parametric equations describes a group of quantities as functions of a common independent variable, called a parameter. The set of parametric equations can alternatively be represented as an equivalent vector function which can simplify later calculations. Comparing the values of the label or amino acid concentrations of three or more labelled amino acid types measured in the sample to the known values of the label or amino acid concentrations of the same three or more amino acid types provided as a function of (unknown) concentration of each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest allows identification of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest within the sample, and simultaneous identification of the concentration and / or amount of that protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest within the sample. Optionally, this can be achieved by creating a vector function, or set of parametric equations, describing any protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest.

[0043] In some embodiments, the set of parametric equations provides the signature of amino acid concentrations that would be measured for three or more amino acid types in the protein, peptide, oligopeptide, polypeptide, or protein complex of interest. The number of parametric equations describing the protein, peptide, oligopeptide, polypeptide, or protein complex of interest is the number of three or more amino acid types labelled and measured in the sample. The parametric equations describe the amino acid concentrations of each of three or more amino acid types labelled and measured in the sample of the protein, peptide, oligopeptide, polypeptide, or protein complex of interest as a function of concentration, t. Set of parametric equations 1 is: <menclose notation="box"> p i t = a 1 t , a 2 t , ⋯ a n t , ∀ t ≥ 0 < / menclose> wherein p i are the amino acid concentrations provided for protein, peptide, oligopeptide, polypeptide, or protein complex of interest i as a function of its concentration t, a 1 is the number of amino acids of amino acid type 1 in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, a 2 is the number of amino acids of amino acid type 2 in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, a n is the number of amino acids of amino acid type n in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, t is the total molar concentration of the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, and there are n parametric equations in the set for the n amino acid types labelled and measured in the sample. In some embodiments, t is defined for all values of t greater than or equal to 0, <menclose notation="box"> ∀ ≥ 0 < / menclose>. In other embodiments, t is provided between a lower (c 1 ) and upper (c 2 ) limit of a concentration range (<menclose notation="box"> ∀ t ∈ c 1 ≤ t ≥ c 2 < / menclose>).

[0044] Set of parametric equations 1 can alternatively be collectively described as a vector function, describing the same reference line or reference curve. The representations are interchangeable. In this representation, vector function 1 is: <menclose notation="box"> p i t = 0 , 0 ,⋯ 0 + a 1 t , a 2 t , ⋯ a n t , ∀ t ≥ 0 < / menclose> where p i are the amino acid concentrations provided for protein, peptide, oligopeptide, polypeptide, or protein complex of interest i as a function of concentration t, <menclose notation="box"> 0 , 0 ⋯ 0 < / menclose>, is the origin, a 1 is the number of amino acids of amino acid type 1 in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, a 2 is the number of amino acids of amino acid type 2 in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, a n is the number of amino acids of amino acid type n in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, t is the total molar concentration of the protein, peptide, polypeptide, oligopeptide, or protein complex of interest which is defined for all values of t greater than or equal to 0 (<menclose notation="box"> ∀ t ≥ 0 < / menclose>). In alternative embodiments, t is provided between a lower (c 1 ) and upper (c 2 ) limit of a concentration range (<menclose notation="box"> ∀ t ∈ c 1 ≤ t ≥ c 2 < / menclose>), and the vector begins at the amino concentrations of the lower bound of the concentration range, <menclose notation="box"> a 1 c 1 , a 2 c 1 ⋯ a n c 1 < / menclose>.

[0045] For example, there are 2 proteins of interest and 1 protein complex of interest. The first protein of interest is BSA. The K (a 1 ), C (a 2 ), and W (a 3 ) amino acid types are labelled and measured in the sample. a 1 = 59, a 2 = 35, and a 3 = 2 because there are 59, 35, and 2 amino acids of the K, C, and W amino acid types within the protein sequence of BSA, respectively. The vector function providing the amino acid concentrations as a function of protein concentration of BSA is <menclose notation="box"> p BSA t = 0 0 0 + 59 t , 35 t , 2 t , ∀ t ≥ 0 < / menclose>

[0046] Hen egg white lysozyme (LYZ) is the second protein of interest. a 1 = 6, a 2 = 8, and a 3 = 6 because there are 6, 8, and 6 amino acids of the K, C, and W amino acid types within the protein sequence of LYZ, respectively. The vector function providing the amino acid concentrations as a function of protein concentration of LYZ is <menclose notation="box"> p LYZ t = 0 0 0 + 6 t , 8 t , 6 t , ∀ t ≥ 0 < / menclose>

[0047] Transthyretin is the protein complex of interest. a 1 = 32, a 2 = 4, and a 3 = 8 because there are 32, 4, and 8 amino acids of the K, C, and W amino acid types within all of the protein sequences comprising the protein complex of interest (the number of amino acids of each of the 4 subunits of the protein complex are summed). The vector function providing the amino acid concentrations as a function of concentration of transthyretin (TTR) is <menclose notation="box"> p TTR t = 0 0 0 + 32 t , 4 t , 8 t , ∀ t ≥ 0 < / menclose>

[0048] The vector equation for BSA provides a reference line for BSA in n dimensional space (3-dimensional space, because 3 types of amino acids are labelled and measured in the experiment), the vector equation for LYZ provides a reference line for LYZ in n dimensional space, and the vector equation for TTR provides a reference line for TTR in n dimensional space. These vector equations and corresponding reference lines are plotted in Figure 2, along with a sample point. To identify the presence and / or concentration and / or amount of one of these proteins or protein complexes of interest within the sample, the distance between the sample point and each of the reference lines provided for BSA, LYZ, and TTR are calculated and compared.

[0049] Previously, methods for the identification of a whole proteome or subproteome within a sample have not been available. It has been required to identify a proteome or subproteome within a sample via separation of the proteins, peptides, oligopeptides, polypeptides, and protein complexes comprising the proteome or subproteome within the sample followed by sequential identification of each protein, peptide, oligopeptide, polypeptide, and protein complex within the proteome or subproteome.

[0050] It has been discovered that it is not necessary to separate a proteome, subproteome, or other mixture of proteins within a sample in order to identify the proteome, subproteome, or other mixture and determine the concentration or amount of the proteome, subproteome, or other mixture. It has been discovered that it is not necessary to identify every protein within a proteome, subproteome or other mixture in order to identify and determine the concentration or amount of the proteome, subproteome, or other mixture. Instead, only a single measurement of the amino acid concentration, value of the label, or number of amino acids of three or more amino acid types of a proteome, subproteome, or other mixture contained within the sample has to be made.

[0051] It has been discovered that a proteome or subproteome within a sample can be alternatively thought of as an average protein sequence whose numbers of amino acids are a weighted mean of the numbers of amino acids of each protein, peptide, oligopeptide, polypeptide, or protein complex sequence within the proteome or subproteome, and whose concentration within the sample is the total molar protein concentration of all proteins, peptides, oligopeptides, polypeptides, or protein complexes which comprise the proteome or subproteome. An unseparated proteome or subproteome within a sample can be identified and quantified in this manner, because it has been discovered that these signatures are unique for each proteome and subproteome. The order of amino acids within this average protein sequence is not calculated, and the number of amino acids of three or more amino acid types within every such average protein sequence is unique for all proteomes and subproteomes. For example, the number of amino acids of three or more amino acid types within every average protein sequence is unique for all known bacterial proteomes and all known viral proteomes (Figure 3). This is demonstrated for the 7581 known bacterial reference proteomes and the 9377 known viral reference proteomes. A reference proteome is a complete proteome. Therefore, all known bacterial proteomes and all known viral proteomes have a distinct signature which can easily be detected within a sample using the methods of the invention without separating proteins, peptides, oligopeptides, polypeptides, or protein complexes which comprise a proteome from one another. This is a counterintuitive result, because while it would be expected that the number of amino acids of two or more amino acid types of proteins, oligopeptides, polypeptides, and protein complexes within a proteome would vary according to a distribution, it would be expected that the mean of the distribution for each proteome would cluster around single values dictated by biological function. Also, the mean number of amino acids of two or more amino acid types across proteomes does not follow the trend x = y = z, suggesting that this variability cannot be accounted for by differences in the mean length of protein, oligopeptide, polypeptide, and protein complex sequences across proteomes.

[0052] Current methods of diagnosing infection, such as SARS-CoV-2 infection, rely on the reverse transcription polymerase chain reaction (RT-PCR) for (generally qualitative) determination of SARS-CoV-2 RNA within patient samples. However, these tests have a 30% false negative rate, which has significant consequences for patient care, infection control, and modeling.

[0053] In addition to providing a new approach for the rapid diagnosis of any infection, the methods of the invention can be applied to the identification of the presence and / or concentration and / or amount of a disease-associated subproteome of interest within a patient sample. For example, the subproteomic signature of type 1 diabetes mellitus can be identified and quantified in saliva. In some embodiments, the subproteomic signature of human ovarian cancer, human pancreatic cancer, human prostate cancer or human colorectal cancer can be identified and quantified in blood plasma samples. In some embodiments, the subproteomic signature of human bladder cancer, human prostate cancer or human renal cancer can be identified and quantified in urine samples.

[0054] In some embodiments, when one or more subproteomes or proteomes are of interest, then the number of amino acids of a particular amino acid type is the weighted mean number of amino acids of a particular amino acid type across all of the proteins in the subproteome or proteome of interest. For example, if the three or more amino acid types labelled in the sample inlcude tryptophan (W) and lysine (K), and the proteome of interest in the sample is the SARS-CoV-2 proteome, then the weighted mean number of tryptophan (W) and the weighted mean number of lysine (K) amino acids in the average amino acid sequence of all of the proteins of the SARS-CoV-2 proteome is calculated from the amino acid sequences of the SARS-CoV-2 proteome as 11.3 W and 60.6 K.

[0055] It has been discovered that any proteome or subproteome of interest can be described by a set of parametric equations. In some embodiments, the parametric equations provide a signature of amino acid concentrations that would be measured for three or more amino acid types in the proteome or subproteome. The set of parametric equations depending on the common parameter of concentration is set of parametric equations 2 and takes the form: <menclose notation="box"> p i t = w 1 t , w 2 t , ⋯ w n t , ∀ t ≥ 0 < / menclose> where p i are the amino acid concentrations provided for proteome or subproteome of interest i as a function of proteome / subproteome concentration t (wherein the proteome / subproteome concentration is the total molar concentration of all proteins, peptides, oligopeptides, polypeptides, and protein complexes comprising proteome or subproteome of interest p i ), w 1 is the weighted mean number of amino acids of amino acid type 1 in the proteome or subproteome of interest, w 2 is the weighted mean number of amino acids of amino acid type 2 in the proteome or subproteome of interest, w n is the weighted mean number of amino acids of amino acid type n in the proteome or subproteome of interest, t is the proteome or subproteome concentration (wherein the proteome or subproteome concentration is the total molar concentration of all proteins, peptides, oligopeptides, polypeptides, and protein complexes comprising proteome or subproteome of interest, p i ). In some embodiments, the proteome or subproteome concentration t is defined for all values of t greater than or equal to 0. There are n parametric equations in the set for the n amino acid types labelled and measured in the sample.

[0056] The unique signature of amino acid concentrations provided for a proteome or subproteome of interest can be equivalently described using vector function 2: <menclose notation="box"> p i t = 0 , 0 ,⋯ 0 + w 1 t , w 2 t , ⋯ w n t , ∀ t ≥ 0 < / menclose> where p i are the amino acid concentrations provided for proteome or subproteome of interest i as a function of the concentration, t, of the proteome or subproteome, <menclose notation="box"> 0 , 0 ,⋯ 0 < / menclose> is the origin, w 1 is the weighted mean number of amino acids of amino acid type 1 in the proteome or subproteome of interest, w 2 is the weighted mean number of amino acids of amino acid type 2 in the proteome or subproteome of interest, w n is the number of amino acids of amino acid type n in the proteome or subproteome of interest, and t is the proteome or subproteome concentration (wherein the proteome or subproteome concentration is the total molar concentration of all proteins, peptides, oligopeptides, polypeptides, and protein complexes comprising proteome or subproteome of interest, p i ). In some embodiments, the proteome or subproteome concentration t is defined for all values of t greater than or equal to 0.

[0057] In some embodiments, the mean number of amino acids of each of the same three or more amino acid types as were labelled and measured in the sample in the proteome or subproteome of interest is the weighted mean number of amino acids of each of the same three or more amino acid types as were labelled and measured in the sample. In some embodiments, the weights of the weighted mean are provided by the proportion of that protein sequence within the total number of protein sequences in the proteome or subproteome of interest. For example, the weighted mean number of tryptophan (W) amino acids per proteome is equal to a linear combination of the number of tryptophan amino acids per protein sequence multiplied by the proportion of that protein sequence within all protein sequences comprising the proteome or subproteome of interest, and the weighted number of lysine (K) amino acids per proteome is equal to a linear combination of the number of tryptophan amino acids per protein sequence multiplied by the proportion of that protein sequence within all protein sequences comprising the proteome or subproteome of interest.

[0058] The amino acid concentrations measured for three or more labelled amino acid types in the sample are compared to the amino acid concentrations of the same three or more amino acid types provided for one or more proteomes or subproteomes of interest. This allows identification of the sample as one of the proteomes or subproteomes of interest as well as determination of the concentration or amount of the proteome or subproteome of interest present within the sample.

[0059] In some embodiments, the concentration of each of three or more amino acid types is the concentration of that labelled amino acid type of each protein, peptide, oligopeptide, polypeptide, or protein complex of interest. In some embodiments, the concentration of each of the three or more amino acid types of each proteome or subproteome of interest is the total concentration of that labelled amino acid type across the proteins in the proteome or subproteome of interest. This is because the concentration of the amino acid type is equal to the mean number of amino acids per sequence in the proteome multiplied by the total protein concentration of the proteome.

[0060] Frequently, the molar protein concentration of an unknown sample is not known, because if standard methods in the art are used to determine the absorption (A 280 ) or mass protein concentration of the sample, this cannot be converted to the molar protein concentration of the sample unless the molecular weight of the sample is known, and the molecular weight of the sample is unknown because the identity of the sample is unknown.

[0061] In some embodiments, the molar protein concentration of the sample is known. Let the known molar protein concentration of the sample be the constant SC. Therefore, if protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest p i is present within the sample, then it is present at the molar protein concentration of the sample, so t = SC. The result of this special case is considered using the example of set of parametric equations 1: <menclose notation="box"> p i t = a 1 t , a 2 t , ⋯ a n t , ∀ t ≥ 0 < / menclose> , which simplifies to a point in n dimensional space <menclose notation="box"> p i = a 1 SC , a 2 SC , … , a n SC < / menclose>

[0062] This is no longer a set of parametric equations because it is not a function of a common parameter (independent variable), because the variable t was replaced with the constant SC. In this embodiment, the amino acid concentrations for protein of interest p i instead provide a point in n dimensional space.

[0063] In some embodiments, the amino acid concentrations of each of three or more amino acid types of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest are used to determine the corresponding label values of each of the same three or more amino acid types for the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest with a set of parametric equations.

[0064] This is achieved by incorporating into the parametric equations describing the amino acid concentrations of any protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest a calibration function or calibration factor which converts between the measured label of each amino acid type and the amino acid concentration of each amino acid type.

[0065] In some embodiments, the parametric equations describe the unique signature of the label values (e.g. signals of the label) for the protein, peptide, oligopeptide, polypeptide, or protein complex of interest as a function of its concentration, t, via set of parametric equations 3: <menclose notation="box"> p i t = a 1 f 1 t + b 1 , a 2 f 2 t + b 2 , ⋯ a n f n t + b n , ∀ t ≥ 0 < / menclose>

[0066] Where p i are the known values of the label provided for protein, peptide, oligopeptide, polypeptide, or protein complex of interest i as a function of its concentration t, a 1 is the number of amino acids of amino acid type 1 in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, a 2 is the number of amino acids of amino acid type 2 in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, a n is the number of amino acids of amino acid type n in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, b 1 is the background value for amino acid type 1 which is 0 if the measured values of the label in the sample are background-corrected, b 2 is the background value for amino acid type 2 which is 0 if the measured values of the label in the sample are background-corrected, b n is the background value for amino acid type n which is 0 if the measured values of the label in the sample are background-corrected, f 1 is the calibration function or calibration factor for amino acid type 1, f 2 is the calibration function or calibration factor for amino acid type 2, f n is the calibration function or calibration factor for amino acid type n, and t is the molar concentration of the protein, peptide, polypeptide, oligopeptide, or protein complex of interest. There are n parametric equations in the set for the n amino acid types labelled and measured in the sample. In some embodiments, t is defined for all values of t greater than or equal to 0, <menclose notation="box"> ∀ t ≥ 0 < / menclose>. In other embodiments, t is provided between a lower (c 1 ) and upper (c 2 ) limit of a concentration range (∀t ∈ c 1 ≤ t ≥ c 2 ),

[0067] The equations constituting set of parametric equations 3 can equivalently be collectively described as vector function 3: <menclose notation="box"> p i t = b 1 , b 2 , ⋯ b n + a 1 f 1 t , a 2 f 2 t , ⋯ a n f n t , ∀ t ≥ 0 < / menclose>

[0068] Where p i are the known values of the label provided for protein, peptide, oligopeptide, polypeptide, or protein complex of interest i as a function of its concentration t, b 1 is the background value for amino acid type 1 which is 0 if the measured values of the label in the sample are background-corrected, b 2 is the background value for amino acid type 2 which is 0 if the measured values of the label in the sample are background-corrected, b n is the background value for amino acid type n, which is 0 if the measured values of the label in the sample are background-corrected, a 1 is the number of amino acids of acid type 1 in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, a 2 is the number of amino acids of amino acid type 2 in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, a n is the number of amino acids of amino acid type n in the protein, peptide, polypeptide, oligopeptide, or protein complex of interest, f 1 is the calibration function or calibration factor for amino acid type 1, f 2 is the calibration function or calibration factor for amino acid type 2, f n is the calibration function or calibration factor for amino acid type n, and t is the molar protein concentration of the protein, peptide, polypeptide, oligopeptide, or protein complex of interest. In some embodiments, t is defined for all values of t greater than or equal to 0 (<menclose notation="box"> ∀ t ≥ 0 < / menclose>). In alternative embodiments, t is provided between a lower (c 1 ) and upper (c 2 ) limit of a concentration range (<menclose notation="box"> ∀ t ∈ c 1 ≤ t ≥ c 2 < / menclose>), and the vector begins at the values of the label of the lower bound of the concentration range, <menclose notation="box"> a 1 f 1 c 1 , a 2 f 2 c 1 , ⋯ a n f n c 1 < / menclose>.

[0069] In other embodiments, the parametric equations describing the unique signature of the label values (e.g. signal of the label) for a proteome or subproteome of interest at any concentration, t, is set of parametric equations 4: <menclose notation="box"> p i t = w 1 f 1 t + b 1 , w 2 f 2 t + b 2 , ⋯ w n f n t + b n , ∀ t ≥ 0 < / menclose> where p i are the known values of the label provided for proteome or subproteome of interest i as a function of its concentration t, w 1 is the weighted mean number of amino acids of amino acid type 1 in the proteome or subproteome of interest, w 2 is the weighted mean number of amino acids of amino acid type 2 in the proteome or subproteome of interest, w n is the weighted mean number of amino acids of amino acid type n in the proteome or subproteome of interest, b 1 is the background value for amino acid type 1 which is 0 if the measured values of the label in the sample are background-corrected, b 2 is the background value for amino acid type 2 which is 0 if the measured values of the label in the sample are background-corrected, b n is the background value for amino acid type n which is 0 if the measured values of the label in the sample are background-corrected, f 1 is the calibration function or calibration factor for amino acid type 1, f 2 is the calibration function or calibration factor for amino acid type 2, f n is the calibration function or calibration factor for amino acid type n, and t is the molar concentration of the proteome or subproteome of interest. There are n parametric equations in the set for the n amino acid types labelled and measured in the sample. In some embodiments, t is defined for all values of t greater than or equal to 0 (<menclose notation="box"> ∀ t ≥ 0 < / menclose>.

[0070] The set of parametric equations in this embodiment can alternatively be collectively described using vector function 4: <menclose notation="box"> p i t = b 1 , b 2 , ⋯ b n + w 1 f 1 t , w 2 f 2 t , ⋯ w n f n t , ∀ t ≥ 0 < / menclose> where p i are the known values of the label provided for proteome or subproteome of interest i as a function of its concentration t, b 1 is the background value for amino acid type 1 which is 0 if the measured values of the label in the sample are background-corrected, b 2 is the background value for amino acid type 2 which is 0 if the measured values of the label in the sample are background-corrected, b n is the background value for amino acid type n which is 0 if the measured values of the label in the sample are background-corrected, w 1 is the weighted mean number of amino acids of amino acid type 1 in the proteome or subproteome of interest, w 2 is the weighted mean number of amino acids of amino acid type 2 in the proteome or subproteome of interest, w n is the weighted mean number of amino acids of amino acid type n in the proteome or subproteome of interest, f 1 is the calibration function or calibration factor for amino acid type 1, f 2 is the calibration function or calibration factor for amino acid type 2, f n is the calibration function or calibration factor for amino acid type n, and t is the molar concentration of the proteome or subproteome of interest. In some embodiments, t is defined for all values of t greater than or equal to 0.

[0071] Therefore, it has been discovered that a set of parametric equations or a vector function can be constructed for any protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest based on the amino acid sequence or sequences of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest alone, describing the unique signatures of the label values (e.g. signals) or amino acid concentrations of three or more amino acid types of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest as a function of concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. For example, if only the label of W and Y has been measured in the sample, and it has not been converted into the amino acid concentration or number of W and Y amino acid types in the sample, then the number of W and Y amino acids in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is converted into the corresponding known label value of W and Y as a function of the unknown concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. This allows the measured label of W and Y in the sample to be compared to the known label value of W and Y in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest, and determination of the presence and / or concentration and / or amount of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest in the sample. In some embodiments, no calculations are required on the signals measured for the sample.

[0072] It was discovered that the vector form of the reference line or reference curve for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest allows direct calculation of the concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest which provides the known values of the label or amino acid concentrations of the three or more amino acid types closest (i.e. the distance between the sample point and the reference line is minimized) to the corresponding three or more amino acid types labelled and measured in the sample.

[0073] This is achieved by finding the dot product of the direction of the reference line with the vector between the sample point and any point on the reference line, setting the dot product equal to 0, and solving for the concentration of the reference line which provides a perpendicular line between the sample point and the reference line. A dot product is a scalar value that represents the angular relationship between two vectors A and B i.e. A · B = |A| * |B| * cos θ where the values |A| and |B| represent the lengths of vectors A and B respectively, and θ is the angle between the two vectors. If A and B are perpendicular (i.e. at 90 degrees to each other) then the dot product will be zero, because cos 90° will be zero. This distance between the sample point and the reference line is calculated, and if this distance is less than or equal to an error margin, then the protein, peptide, oligopeptide, polypeptide, protein complex, proteome, or subproteome of interest is identified as being present at the protein concentration on the reference line which provided the minimum distance.

[0074] In some embodiments, if the sample point is less than or equal to an error margin or distance threshold from more than one protein, peptide, oligpeptide, polypeptide, protein complex, subproteome, or proteome of interest, then a mixture of proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is identified in the sample. If a component within the mixture comprises a larger proportion of the mixture, then its signature will have a greater effect on the signature of the sample than will the signature of a component which comprises a smaller proportion within the mixture. The proportion of components within the mixture is also available using the methods of the invention. The proportion of each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome within the mixture is calculated by comparing the distances between the sample and each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome identified as being present in the sample, where a smaller distance indicates a larger proportion of the component within the mixture. In some embodiments, the distances calculated from the sample point to the reference line for each identified component of the mixture are compared. It was discovered that the proportion of each component within the mixture is determined from the inverse of the normalized distances for each identified component of the mixture. The maximum distance for all identified components is calculated, and this is divided by the distance for each identified component. In some embodiments, the proportion of an identified component within the mixture is calculated by dividing its inverse normalized distance by the sum of the inverse normalized distances from all components within the mixture.

[0075] The methods of the present invention do not require the order (i.e. position) of the amino acids within an amino acid sequence to be determined in order to identify the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest in the sample. The methods of the present invention do not require the sequence of amino acids within proteins in the sample to be determined in order to identify the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest in the sample.

[0076] The methods of the invention can provide a reference for a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome or proteome of interest which is described algebraically using the formulas disclosed herein. There is a variable, which is protein concentration, in the reference. The reference provides the amino acid concentrations or fluorescence intensities which would be measured for any concentration of protein, peptide, oligopeptide, polypeptide, protein complex, subproteome or proteome of interest. This feature makes it possible to quantify the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome or proteome of interest when it is identified. Hence, the methods disclosed herein provide a quantitative technique.BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The following Figures that only refer to labelling two amino acid types within a sample, are not according to the invention and are present for illustration purposes only. Embodiments of the invention are described below with reference to the accompanying drawings, in which: Figure 1 shows a schematic drawing illustrating how the unique signatures calculated for Protein-A of interest, Protein-B of interest, Protein-C of interest, and Protein-D of interest vary as a function of the protein concentration of each protein of interest. Reference vectors are provided for each protein of interest, and each point on the reference vector corresponds to a unique protein concentration of the protein of interest (e.g. 1 µM, filled circle). The shortest distance from the Sample point (open square) to each reference line is calculated, identifying the presence of Protein-B of interest in the Sample; the concentration of Protein-B of interest in the sample is the protein concentration of Protein-B of interest which provided the shortest distance (e.g. 0.5 µM). Figure 2 shows reference lines in n-dimensional space. Set of parametric equations 1 provides the following reference lines for BSA, LYZ, and TTR. The sample point is shown with an open circle. The methods of the invention include determining the presence and / or concentration and / or amount of the proteins / protein complexes of interest in the sample based on a comparison of the distance between the sample point and each reference line. Figure 3 shows the unique signatures for pathogenic proteomes. (a) All 7581 bacterial reference proteomes analysed have a unique signature of known label values, amino acid concentrations, or mean number of amino acids across all proteins in the bacterial reference proteome. (b) Zoomed image showing a wide distribution of the mean number of the number of amino acids of two or more amino acid types within every average protein sequence. (c) All 9377 viral reference proteomes analysed have a unique signature of known label values, amino acid concentrations, or mean number of amino acids across all proteins in the viral reference proteome. (d) All 16958 bacterial and viral reference proteomes analysed have a unique signature of known label values, amino acid concentrations, or mean number of amino acids across all proteins in the bacterial or viral reference proteome. This enables the identification of a whole proteome in a sample without separation. Figure 4 shows analysis of the probability distribution of leading digits in a set of numbers according to Benford's law shows that amino acid types in the human plasma proteome follow the expected distribution. Figure 5 shows analysis of the probability distribution of leading digits in a set of numbers according to Benford's law shows that mean numbers of amino acids across proteins, peptides, oligopeptides, polypeptides, and protein subunits in viral proteomes deviate from the expected distribution, suggesting increased variability in this dataset relative to human proteomes. Figure 6 shows analysis of the probability distribution of leading digits in a set of numbers according to Benford's law shows that mean numbers of amino acids across proteins, peptides, oligopeptides, polypeptides, and protein subunits in bacterial proteomes deviate from the expected distribution, suggesting increased variability in this dataset relative to human proteomes. Figure 7 shows identifying the order of amino acids within a protein sequence within the human proteome is inefficient compared to identifying only the number of amino acids within a protein sequence. Identifying the order of two types of amino acids within a protein sequence adds no additional information to identifying the order of one type of amino acid within a protein sequence. Figure 8 shows demonstration of the effect of constraining the reference line to known protein concentration ranges within the human plasma proteome. (a) Reference lines for all 3263 proteins, peptides, oligopeptides, polypeptides, and protein complexes within the human plasma proteome. (b) Bounded reference lines for all 3263 proteins, peptides, oligopeptides, polypeptides, and protein complexes within the human plasma proteome, wherein the reference lines are bounded by the known concentration ranges of these proteins, peptides, oligopeptides, polypeptides, and protein complexes within the human plasma proteome. Figure 9 shows the occurrences of references referring to more than one protein of interest was quantified across the human plasma proteome for various combinations of amino acid types (C and W, K and W, K and Y, K and S, K and P, L and S, L and K, E and L, G and L, C K and W, C K and Y, L K and S, E G and K, E G and S, R E P and T, and Q L K and V - with and without protein concentration information, accessible via the methods of the invention, compared to known protein concentration bounds. Figure 10 shows when two amino acid types are labelled and compared, without application of any bounds or constraints on the protein concentration or other classification, all references are distinguishable and map uniquely to proteins of interest within most of the clinically relevant proteomes and subproteomes considered (SARS-CoV-2, HIV, Epstein-Barr, Glioma) and do not correspond to multiple proteins of interest within the clinically relevant proteomes and subproteomes. Figure 11 shows comparing the information content provided by all combinations of two amino acid types to the uniqueness of references for protein sequences within the (a) human plasma proteome and (b) human salivary proteome. Figure 12 shows that all reference bacterial proteomes (7581 reference proteomes) have a mean number of amino acids within two amino acid types across proteins in their proteomes that is distinct from all other mean numbers of amino acids within two amino acid types across proteins all other proteomes. Figure 13 shows that for the labelling of only two amino acid types within a proteome of interest, bacterial and viral proteomes cluster together according to their lineage. Here the labeling of K and W amino acid types is provided, showing clustering within the orders: Corynebacteriaceae, Legionellales, Bacillales, Streptomycetaceae, and Mycoplasmataceae. Figure 14 describes the treatment for an unknown mixture of proteins. The identity of the mixture is unknown, and the protein concentration of the mixture is unknown. Figure 15 shows that hydrodynamic radius cannot be predict based on protein sequences alone, because state-of-the art scaling methods still require knowledge of whether a protein is folded or unfolded, and do not account for partial intrinsic disorder. Figure 16 schematics showing the reaction of (a) Tryptophan (W), (b) Tyrosine (Y), (c) Reduced Cysteine (C R ), (d) Cysteine (C), and (e) Lysine (K) amino acid types with fluorogenic dyes, or molecules which becomes fluorescent upon reaction with the indicated amino acid type. Figure 17 shows comparison of Patient samples to (a) C and K, (b) C and W, and (C) K and W SARS-CoV-2 and Influenza A reference lines. Figure 18 shows a calibration curve for conversion of background-corrected fluorescence intensity from the K amino acid type K F.I. in arbitrary units (AU) to amino acid concentration of the K amino acid type [K] in µM. Nonlinear regression revealed a provided a polynomial fit for the calibration curve with R 2< = 0.9987. Figure 19 shows a calibration curve for conversion of background-corrected fluorescence intensity from the C amino acid type C F.I. in arbitrary units (AU) to amino acid concentration of the C amino acid type [C] in µM. Nonlinear regression revealed a provided a polynomial fit for the calibration curve with R 2< = 0.9886. Figure 20 shows a calibration curve for conversion of background-corrected fluorescence intensity from the W amino acid type W F.I. in arbitrary units (AU) to amino acid concentration of the W amino acid type [W] in µM. Nonlinear regression revealed a provided a polynomial fit for the calibration curve with R 2< = 0.9886. Figure 21 shows that when the mean measured amino acid concentrations across the three technical replicates of each experimentally measured patient PPP sample are plotted in N-dimensional space (4-dimensional space), the data takes on a line in N-dimensional space as predcted by the concepts of the invention. This conceptual line was illustrated by drawing a line through the data set. To calculate the actual position and equation of the reference line defining the PPP proteome of interest, the K, C, W, and Y components of the vector function defining the PPP proteome of interest were calculated experimentally in the following figures. Figure 22 shows how the coefficient (direction) of the K component of the experimental reference line was calculated for the PPP and PRP proteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type K are plotted against the measured total protein concentrations in µg / mL for each proteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 23 shows how the coefficient (direction) of the C component of the experimental reference line was calculated for the PPP and PRP proteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type C are plotted against the measured total protein concentrations in µg / mL for each proteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 24 shows how the coefficient (direction) of the W component of the experimental reference line was calculated for the PPP and PRP proteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type W are plotted against the measured total protein concentrations in µg / mL for each proteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 25 shows how the coefficient (direction) of the Y component of the experimental reference line was calculated for the PPP and PRP proteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type Y are plotted against the measured total protein concentrations in µg / mL for each proteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 26 shows how the coefficient (direction) of the K component of the experimental reference line was calculated for the PPP_50 and PRP_50 subproteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type K are plotted against the measured total protein concentrations in µg / mL for each subproteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 27 shows how the coefficient (direction) of the C component of the experimental reference line was calculated for the PPP_50 and PRP_50 subproteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type C are plotted against the measured total protein concentrations in µg / mL for each subproteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 28 shows how the coefficient (direction) of the W component of the experimental reference line was calculated for the PPP_50 and PRP_50 subproteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type W are plotted against the measured total protein concentrations in µg / mL for each subproteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. In the subsequent 8 figures, how the coefficient (direction) of the components of the experimental reference lines based on the common parameter of molar, rather than mass, protein concentration is explained. Figure 29 shows how the coefficient (direction) of the K component of the experimental reference line was calculated for the PPP and PRP proteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type K are plotted against the measured total protein molar concentrations in µM for each proteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 30 shows how the coefficient (direction) of the C component of the experimental reference line was calculated for the PPP and PRP proteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type C are plotted against the measured total protein molar concentrations in µM for each proteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 31 shows how the coefficient (direction) of the W component of the experimental reference line was calculated for the PPP and PRP proteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type W are plotted against the measured total protein molar concentrations in µM for each proteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 32 shows how the coefficient (direction) of the Y component of the experimental reference line was calculated for the PPP and PRP proteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type Y are plotted against the measured total protein molar concentrations in µM for each proteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 33 shows how the coefficient (direction) of the K component of the experimental reference line was calculated for the PPP_50 and PRP_50 subproteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type K are plotted against the measured total protein molar concentrations in µM for each subproteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 34 shows how the coefficient (direction) of the C component of the experimental reference line was calculated for the PPP_50 and PRP_50 subproteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type C are plotted against the measured total protein molar concentrations in µM for each subproteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 35 shows how the coefficient (direction) of the W component of the experimental reference line was calculated for the PPP_50 and PRP_50 subproteomes of interest. The measured amino acid molar concentrations in µM of the amino acid type W are plotted against the measured total protein molar concentrations in µM for each subproteome of interest and a linear regression was performed. The linear regression was constrained to pass through the origin. Figure 36 shows the mean measured amino acid concentrations across the three technical replicates of each patient sample (stars) and the theoretical reference line (solid line). The close agreement between the experimentally measured dataset and predicted reference line illustrate the robustness of the approach disclosed herein whereby any proteome or subproteome of interest can be described algebraically by a single reference which is a vector function of a common parameter of total protein concentration. Figure 37 shows the amino acid concentrations in µM of the amino acid type K versus amino acid concentrations in µM of the amino acid type C, for both PPP and PRP proteomes of interest. This dataset was partitioned into a training set and a testing set, the training set was used to train a classifier to identify the proteome of interest of a patient sample based on its measured concentratinos of the K and C amino acid types. Figure 38 shows the predictions of the trained classifier explained in Figure 37. There are no incorrect predictions shown because 100% of its predictinos were correct. Figure 39 shows a 100% percentage of accuracy (true versus predicted class using a Fine K-Nearest Neighbor, KNN classifier of PPP vs PRP proteome identification using only the amino acid concentrations calculated from the measured values of the label of two labeled amino acid types: K and C. Figure 40 shows that the high classification sensitivities and specificites are robust to the type of classifier used. For example, an 100% percentage of accuracy (true versus predicted class using a Bagged Decision Tree classifier) of PPP vs PRP proteome identification using just the two amino acid types K and C is shown. Additionally no optimization or hyperparameter tuning was required to achieve this level (100% accuracy) of classifier performance based on the amino acid concentrations calculated from the measured values of the label of two labeled amino acid types: K and C. Figure 41 shows a 100% Positive Predictive Value (true versus predicted class using a Bagged Decision Tree classifier) of PPP vs PRP proteome identification using just the two amino acid types K and C. Figure 42 shows the K coefficient of the experimental reference line for the PPP and PRP proteomes of interest for every individual male and female patient plotted as a function of patient age. There was there was no impact of patient gender or age on the coefficient of the experimental proteomic reference lines calculated for each patient. This confirms that the proteomic signatures measured using the methods of the present invention describe any patient population and are specifically not affected by gender or age. This result confirms that the methods of the invention are robust to individual patient variations and that healthy patients exhibt a single identifying proteomic signature. Figure 43 shows the C coefficient of the experimental reference line for the PPP and PRP proteomes of interest for every individual male and female patient plotted as a function of patient age. There was there was no impact of patient gender or age on the coefficient of the experimental proteomic reference lines calculated for each patient. This confirms that the proteomic signatures measured using the methods of the present invention describe any patient population and are specifically not affected by gender or age. This result confirms that the methods of the invention are robust to individual patient variations and that healthy patients exhibt a single identifying proteomic signature. Figure 44 shows the W coefficient of the experimental reference line for the PPP and PRP proteomes of interest for every individual male and female patient plotted as a function of patient age. There was there was no impact of patient gender or age on the coefficient of the experimental proteomic reference lines calculated for each patient. This confirms that the proteomic signatures measured using the methods of the present invention describe any patient population and are specifically not affected by gender or age. This result confirms that the methods of the invention are robust to individual patient variations and that healthy patients exhibt a single identifying proteomic signature. Figure 45 shows the Y coefficient of the experimental reference line for the PPP and PRP proteomes of interest for every individual male and female patient plotted as a function of patient age. There was there was no impact of patient gender or age on the coefficient of the experimental proteomic reference lines calculated for each patient. This confirms that the proteomic signatures measured using the methods of the present invention describe any patient population and are specifically not affected by gender or age. This result confirms that the methods of the invention are robust to individual patient variations and that healthy patients exhibt a single identifying proteomic signature. Figure 46 shows the K coefficient of the experimental reference line for the PPP_50 and PRP_50 subproteomes of interest for every individual male and female patient plotted as a function of patient age. There was there was no impact of patient gender or age on the coefficient of the experimental subproteomic reference lines calculated for each patient. This confirms that the subproteomic signatures measured using the methods of the present invention describe any patient population and are specifically not affected by gender or age. This result confirms that the methods of the invention are robust to individual patient variations and that healthy patients exhibt a single identifying subproteomic signature. Figure 47 shows the C coefficient of the experimental reference line for the PPP_50 and PRP_50 subproteomes of interest for every individual male and female patient plotted as a function of patient age. There was there was no impact of patient gender or age on the coefficient of the experimental subproteomic reference lines calculated for each patient. This confirms that the subproteomic signatures measured using the methods of the present invention describe any patient population and are specifically not affected by gender or age. This result confirms that the methods of the invention are robust to individual patient variations and that healthy patients exhibt a single identifying subproteomic signature. Figure 48 shows the W coefficient of the experimental reference line for the PPP_50 and PRP_50 subproteomes of interest for every individual male and female patient plotted as a function of patient age. There was there was no impact of patient gender or age on the coefficient of the experimental subproteomic reference lines calculated for each patient. This confirms that the subproteomic signatures measured using the methods of the present invention describe any patient population and are specifically not affected by gender or age. This result confirms that the methods of the invention are robust to individual patient variations and that healthy patients exhibt a single identifying subproteomic signature. Figure 49 shows w k , w C , w W , and w Y values calculated from healthy patient mass spectrometry data compared to w k , w C , w W , and w Y values calculated from healthy patient Human Peptide Atlas immunoassay data with both sets of w k , w C , w W , and w Y values calculated using the methods of the present invention. The agreement of these values illustrates that equation 11 robustly performs on abundance data generated from both mass spectrometry and immunoassay, providing a means to build a congruent / unified set of references (vector functions), even though different experimental techniques were employed to generate the underlying data. The provides a framework to build upon existing sources of data. Figure 50 shows the amino acid concentrations in µM for the amino acid types C, W, Y and K in a plotted in an N-dimensional space for ovarian cancer plasma samples, pancreatic cancer plasma samples, colorectal cancer plasma samples and healthy patient plasma (PPP) samples, and that each of these data sets is observed to take on the form of a reference line (which is the function of the common parameter of total protein concentration) as taught herein. Figure 51 shows amino acid concentration in µM of amino acid type K plotted as a function of total molar protein concentration in µM for the ovarian cancer plasma proteome and calculation of the K coefficient (direction) of the ovarian cancer reference line. Figure 52 shows amino acid concentration in µM of amino acid type C plotted as a function of total molar protein concentration in µM for the ovarian cancer plasma proteome and calculation of the C coefficient (direction) of the ovarian cancer reference line. Figure 53 shows amino acid concentration in µM of amino acid type W plotted as a function of total molar protein concentration in µM for the ovarian cancer plasma proteome and calculation of the W coefficient (direction) of the ovarian cancer reference line. Figure 54 shows amino acid concentration in µM of amino acid type Y plotted as a function of total molar protein concentration in µM for the ovarian cancer plasma proteome and calculation of the Y coefficient (direction) of the ovarian cancer reference line. Figure 55 shows amino acid concentration in µM of amino acid type K plotted as a function of total molar protein concentration in µM for the pancreatic cancer plasma proteome and calculation of the K coefficient (direction) of the pancreatic cancer reference line. Figure 56 shows amino acid concentration in µM of amino acid type C plotted as a function of total molar protein concentration in µM for the pancreatic cancer plasma proteome and calculation of the C coefficient (direction) of the pancreatic cancer reference line. Figure 57 shows amino acid concentration in µM of amino acid type W plotted as a function of total molar protein concentration in µM for the pancreatic cancer plasma proteome and calculation of the W coefficient (direction) of the pancreatic cancer reference line. Figure 58 shows amino acid concentration in µM of amino acid type Y plotted as a function of total molar protein concentration in µM for the pancreatic cancer plasma proteome and calculation of the Y coefficient (direction) of the pancreatic cancer reference line. Figure 59 shows amino acid concentration in µM of amino acid type K plotted as a function of total molar protein concentration in µM for the colorectal cancer plasma proteome and calculation of the K coefficient (direction) of the colorectal cancer reference line. Figure 60 shows amino acid concentration in µM of amino acid type C plotted as a function of total molar protein concentration in µM for the colorectal cancer plasma proteome and calculation of the C coefficient (direction) of the colorectal cancer reference line. Figure 61 shows amino acid concentration in µM of amino acid type W plotted as a function of total molar protein concentration in µM for the colorectal cancer plasma proteome and calculation of the W coefficient (direction) of the colorectal cancer reference line. Figure 62 shows amino acid concentration in µM of amino acid type Y plotted as a function of total molar protein concentration in µM for the colorectal cancer plasma proteome and calculation of the Y coefficient (direction) of the colorectal cancer reference line. Figure 63 shows that when the vector function approach described herein as one possible way of determining the presence and / or concentration and / or amount of a proteome or subproteome of interest in a patient sample is carried out, very high sensitivities and specificites are obtained for determination of the presence and absence of colorectal cancer, ovarian cancer, and pancreatic cancer in patient blood plasma. Specifically, as is outlined in the provided confusion matrix, 100% accuracy is achieved for the identification of colorectal cancer and pancreatic cancer from blood plasma, 90% accuracy for the identification of ovarian cancer from blood plasma, and 95% specificity for the correct identification of cancer negative, healthy samples as cancer-negative, healthy samples. Figure 64 shows that the concentration and / or amount of a proteome of interest which are determined as part of the (quantitative) vector function approach described herein are highly accurate, following the line y = x for all proteomes of interest, and allowing for the determination of the concentration and / or amount of a proteome of interest within a patient sample with very low error (only 2% error). Figure 65 shows that the proteomes of interest can also be identified in blood plasma using a machine learning classifier. A linear support vector machine (SVM) classifier was trained on the molar (µM) amino acid concentrations of the K, C, W, and Y amino acid types of patient plasma samples with 25% of the data held out. A 100% positive predictive value and 0% false discovery rate was obtained for each cancer proteome of interest (all cancer patient samples) as well as the healthy proteome of interest (all healthy patient samples). Figure 66 shows that the proteomes of interest can also be identified in blood plasma using a machine learning classifier trained on amino acid concentrations of only three labeled amino acid types. A linear support vector machine (SVM) classifier was trained on the molar (µM) amino acid concentrations of the K, C, and W amino acid types of patient plasma samples with 25% of the data held out. A 100% positive predictive value and 0% false discovery rate was obtained for each cancer proteome of interest (all cancer patient samples) as well as the healthy proteome of interest (all healthy patient samples). Figure 67 shows that the proteomes of interest can also be identified in blood plasma using a machine learning classifier trained on amino acid concentrations of only two labeled amino acid types. A linear support vector machine (SVM) classifier was trained on the molar (µM) amino acid concentrations of only the K and C amino acid types of patient plasma samples with 25% of the data held out. A 100% positive predictive value and 0% false discovery rate was obtained for each cancer proteome of interest (all cancer patient samples) as well as the healthy proteome of interest (all healthy patient samples). Figure 68 shows a confusion matrix indicating 78% accuracy for detecting stage III colorectal cancer using the methods of the invention based on the amount of the K, C, W, and Y amino acid types. Figure 69 shows a confusion matrix indicating 100% positive predictive value for detecting the location of colorectal cancer using the methods of the invention based on the amount of the K, C, W, and Y amino acid types. Figure 70 shows the molar concentration of amino acids in µM of the K, C, W, and Y amino acid types within bladder cancer samples, prostate cancer samples and renal cancer samples measured in urine. Figure 71 shows a positive predictive value false discovery confusion matrix, indicating 100% positive predictive identification and 0% false discovery for the identification of bladder cancer, prostate cancer, and renal cancer from urine samples using the methods of the invention. All included types of cancer (bladder cancer, prostate cancer, and renal cancer) can be correctly identified from urine samples with a true positive rate of 100% and a false negative rate of 0%. DETAILED DESCRIPTION OF THE INVENTION

[0078] The embodiments that only label two amino acid types within the sample, are not according to the invention and are present for illustration purposes only. The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0079] The invention is based on the discovery that it is only necessary to measure the label and / or amino acid concentration or number of amino acids of three or more amino acid types in a sample in order to identify the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest within a sample. It is only necessary to label and measure three or more amino acid types within a sample in order to identify and quantify proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes or proteomes, without the need to sequence the sample. This is because each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome has a unique signature based on the measured label, amino acid concentration and / or number of amino acids of three or more amino acid types in the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome. The measured label and amino acid concentration signature of three or more amino acid types in a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome is unique based on the concentration of that protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome. It is not necessary to identify the order of the amino acids in the sample, for example, by sequencing the sample for the identification of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest in a sample. Identifying the order of amino acids within a protein sequence in a sample adds no additional information when three or more amino acid types are labelled and measured in a sample (Figure 7).

[0080] The methods of the invention described herein can be used to identify the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest in a sample. This is because each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest has a unique signature based on the known label values, amino acid concentrations or number of amino acids of three or more amino acid types in each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. Therefore, the signature of the sample can be compared to the signature of a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest in order to identify the presence and / or concentration and / or amount of a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest in a sample. For example, the SARS-CoV-2 proteome has a unique signature based on the known label values and / or amino acid concentrations and / or number of amino acids of each amino acid type and the concentration of the SARS-CoV-2 proteome compared to the known label values and / or amino acid concentrations and / or number of amino acids of each amino acid type and the concentration of the Influenza A proteome. Therefore, the measured label, amino acid concentration and / or number of amino acids of three or more amino acid types in the sample can be determined, and compared to the known label values and / or amino acid concentrations or number of amino acids of the samethree or more amino acid types in the SARS-CoV-2 proteome and / or the HIV proteome to identify the presence and / or concentration and / or amount of the SARS-CoV-2 proteome and / or the HIV proteome in the sample.

[0081] Previously, strategies for identifying a whole proteome or subproteome of interest within a sample at one time were not available. Strategies for the identification of a pathogen, such as SARS-CoV-2, have relied on the reverse transcription polymerase chain reaction (RT-PCR) for the qualitative detection of nucleic acid from SARS-CoV-2. Current state-of-the art tests have an approximately 30% false negative rate, with significant consequences for infection control. Quantitative information about viral load is not routinely available. As a general alternative, the methods of the present invention are used to identify a whole proteome or subproteome of interest within the sample at one time, for example, for the identification of the presence and / or concentration and / or amount of the SARS-CoV-2 proteome of interest within patient samples.

[0082] The methods of the invention described herein can be used to identify the presence and / or concentration and / or amount of a subproteome or proteome of interest in a sample because each subproteome or proteome of interest has a unique signature based on the known values of the label, amino acid concentrations and / or number of amino acids of three or more amino acid types in each protein, peptide, oligopeptide, polypeptide, and protein complex across the subproteome or proteome of interest. Therefore, the signature of the sample can be compared to the signature of one or more subproteomes or proteomes of interest to identify the presence and / or concentration and / or amount of a subproteome or proteome of interest in a sample. For example, the human plasma proteome has a unique signature based on the mean known label values, amino acid concentrations and / or number of amino acids of each amino acid type compared to the mean known label values, amino acid concentrations and / or number of amino acids of each amino acid type in the human eye proteome. Therefore, the measured label, amino acid concentration and / or number of amino acids of three or more amino acid types in the sample can be determined, and compared to the mean known label values, amino acid concentrations and / or number of amino acids of the same three or more amino acid types in a proteome of interest to identify the presence and / or concentration and / or amount of that proteome in the sample.

[0083] The methods of the invention can be used to identify the presence of a viral proteome in a sample. Each viral proteome has a unique signature based on the mean known label values, amino acid concentrations and / or number of amino acids of three or more amino acid types. Therefore, the mean measured label, amino acid concentration and / or number of amino acids of three or more amino acid types in the sample can be compared to the mean known label values, amino acid concentrations and / or number of amino acids of the same three or more amino acid types of a viral proteome to identify the presence and / or concentration and / or amount of the viral proteome in the sample. In addition, the methods of the invention can be used to identify the viral load of the viral proteome within the sample. For example, each viral proteome has a unique signature based on the mean number of amino acids of three or more amino acid types in each protein across the viral proteome multiplied by the total protein concentration of the viral proteome. Therefore, the amino acid concentration of three or more amino acid types in the sample can be compared to the mean amino acid concentration of the same three or more amino acid types of a viral proteome at one or more protein concentrations to identify the concentration of the viral proteome within the sample.

[0084] Previously, when using solution phase strategies to identify the presence and / or concentration and / or amount of a mixture of proteins, peptides, polypeptides, oligopeptides, subproteomes or proteomes in a sample, it has been necessary to first separate the mixture into its individual protein components. For example, the proteins within a mixture are separated based on size by gel electrophoresis, or based on adsorption of compounds to the adsorbent using a chromatography column, before individual proteins are sequenced. For example, if a mixture contains two proteins; bovine serum albumin and lysozyme, it was previously necessary to separate the mixture into the individual protein components of bovine serum albumin and lysozyme. In contrast, it has been discovered that the methods of the invention described herein can be used to identify the presence and / or concentration and / or amount of a mixture of proteins, peptides, polypeptides, oligopeptides, subproteomes or proteomes in a sample, based on the average signature from the whole mixture, without the need to separate the mixture into the individual components. For example, a mixture that contains bovine serum albumin and lysozyme can be identified without the need to separate the mixture into its individual protein components of bovine serum albumin and lysozyme. This is because it has been discovered that a mixture of proteins, peptides, polypeptides, oligopeptides, subproteomes or proteomes has a unique signature based on the mean number of amino acids of three or more amino acid types across the proteins, peptides, polypeptides, oligopeptides, subproteomes or proteomes in the mixture. For example, a mixture that contains bovine serum albumin and lysozyme has a unique signature based on the mean measured label, amino acid concentration and / or number of amino acids of three or more amino acid types in bovine serum albumin and lysozyme compared to another mixture that contains bovine serum albumin and alpha synuclein, which has a different unique signature based on the mean measured label, amino acid concentration and / or number of amino acids of the same three or more amino acid types in a bovine serum albumin and alpha synuclein mixture. It is not necessary to know the proportion of the components within a mixture in order to identify the presence and / or concentration and / or amount of a mixture within the sample. Instead, the presence of a mixture is identified in the sample when more than one protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is identified in the sample. The signature of the sample is influenced by the signature of each of the components within the mixture. If protein of interest A is identified within the mixture and comprises a higher proportion of the mixture than protein of interest B which has also been identified in the mixture, then the distance between the sample point and the reference line or point for protein of interest A is smaller than the distance between the sample point and the reference line or point for protein of interest B. It was discovered that, conversely, the distances between the sample point and protein of interest A and B can be calculated and compared to determine the proportion of protein of interest A and B in the mixture. Therefore, the signature of the sample can be compared to the signatures of more than one more than one protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest identified as being present in the sample in order to identify the presence and / or concentration and / or amount of such a mixture in the sample.

[0085] The methods of the invention can also be used to identify co-infection of three or more proteomes in a sample, i.e. a mixture of proteomes in a sample. This is because a mixture of proteomes has a unique signature based on the mean known label values, amino acid concentrations and / or number of amino acids of three or more amino acid types in each protein across the mixture of proteomes. Therefore, the measured label value, amino acid concentration and / or number of amino acids of three or more amino acid types in the sample can be determined and compared to the mean known label values, amino acid concentrations and / or number of amino acids of the same three or more amino acid types in more than one proteome of interest. If the presence of more than one proteome of interest is identified within the sample, then a mixture of proteomes of interest is identified within the sample, and the proportion of each proteome within the mixture can be determined as explained above for a mixture of proteins. If proteome of interest A is identified within the mixture and comprises a higher proportion of the mixture than proteome of interest B which has also been identified in the mixture, then the distance between the sample point and the reference line or point for proteome of interest A is smaller than the distance between the sample point and the reference line or point for proteome of interest B. Conversely the distances between the sample point and proteome of interest A and B can be calculated and compared to determine the proportion of proteome of interest A and B in the mixture. Therefore, the signature of the sample can be compared to the signatures of more than one more than one proteome of interest identified as being present in the sample in order to identify the presence and / or concentration and / or amount of such a mixture in the sample. For example, a patient may have a viral and a secondary bacterial infection, or three viral infections. In this case, the bacteria and virus proteomes and the three viral proteomes do not need to be separated from one another before the method of the invention is carried out. This can equally apply to any combination of proteomes, such as bacteria, fungi, protozoa, plant, animal including human, and any combination thereof. The methods of the invention described herein are simple, effortless, and highly efficient and avoid the inherent disadvantages of methods that require known sequencing techniques and / or separation techniques.

[0086] The methods disclosed herein can be applied to any protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, proteome, or mixture of proteins, peptides, polypeptides, oligopeptides, subproteomes and / or proteomes. The methods of the invention simply require the labelling of amino acids of three or more amino acid types and the measuring of these labels. An amino acid type is defined by the R-group specific to each amino acid. The R-group of each type of amino acid is unique. An amino acid type can include modified and / or unmodified amino acids of the 22 proteinogenic amino acids and / or non-proteinogenic or synthetic amino acids.

[0087] The only requirement for the method is that the signature of the three or more amino acid types of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest (e.g reference), or the amino acid sequence and / or any experimental information about post-translational modifications is available. It is not necessary to determine the sequence of amino acids within the sample in order to identify the presence and / or concentration and / or amount of a protein in the sample. In some embodiments, the signature of three or more amino acid types of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is known (e.g. from a database). In some embodiments, the signature of three or more amino acid types of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is determined from the amino acid sequence or sequences of each of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes, of interest as part of the method of the invention. In some embodiments, if the signature of three or more amino acid types of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes, of interest is not known, the amino acid sequence of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest can be used to determine the signature. Alternatively, the signature of three or more amino acid types of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is determined using the methods disclosed herein (e.g. labelling three or more amino acid types, measuring the value of the label, measuring the total protein concentration of the sample via standard methods, and converting the measured label to the number of amino acids of each labelled amino acid type).

[0088] If the one or more of the proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest has one or more amino acid types that include modified amino acids of the amino acid type, then the signature of the modified amino acids of that amino acid type in the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest can be determined. In some embodiments, this is determined from experimental post-translational modification information for the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest. For example, if the amino acid type C has been labelled in the sample and includes the modified cysteine amino acids cysteine disulphide (C D ), then the signature of the number of amino acids of the amino acid type cysteine can include the post-translational modification information for the modified amino acids oxidized cysteine C O . This signature of the sample can be compared to the signature of the known label values, amino acid concentrations, or number of amino acids of modified amino acids (such as oxidized cysteine C D ) in the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest.Definitions

[0089] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein, are incorporated by reference in their entirety unless stated otherwise. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0090] As used herein, the term "presence" refers to the positive identification of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest in a sample.

[0091] As used herein, the term "concentration" refers to the abundance of an entity per unit of volume. An entity can be a molecule, a complex, a monomer within a polymer such as an amino acid contained within a protein chain, or an atom. Mass concentration refers to the mass of an entity per unit of volume. Number concentration refers to the number of molecules of an entity per unit of volume. Molar concentration refers to the number of moles of an entity per unit of volume. The number of moles of entities is the total number of entities contained within the sample divided by the Avogadro constant NA, which is 6.02214076×1023 mol-1. Unless otherwise stated, the term "concentration" refers to the molar concentration of an entity. Reference is frequently made to the "protein, peptide, oligopeptide, polypeptide, or protein complex of interest as a function of concentration, t" or equivalent. This means that t is the concentration of the protein of interest, or, t is the concentration of the peptide of interest, or, t is the concentration of the oligopeptide of interest, or, t is the concentration of the polypeptide of interest, or, t is the concentration of the protein complex of interest. As used herein, in some embodiments, the concentration of a protein complex of interest refers to the concentration of the complex, not the monomer concentration of subunits within the complex. For example, if protein complex of interest α has two subunits, A and B, such that protein complex of interest α can be described with the complex stoichiometry A:B, then the concentration of protein complex α is the concentration of the complex A:B, not the concentration of subunit A plus the concentration of subunit B. Reference is also frequently made to the "subproteome or proteome of interest as a function of concentration, t" or equivalent. The concentration of a subproteome of interest is the total concentration of all proteins, peptides, oligopeptides, polypeptides, and protein complexes which comprise the subproteome of interest. This means that t is the total concentration of all proteins, peptides, oligopeptides, polypeptides, and protein complexes which comprise the subproteome of interest. The concentration of a proteome of interest is the total concentration of all proteins, peptides, oligopeptides, polypeptides, and protein complexes which comprise the proteome of interest. This means that t is the total concentration of all proteins, peptides, oligopeptides, polypeptides, and protein complexes which comprise the proteome of interest. Once the molar concentration of a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest present in the sample has been identified, the mass concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is the molar concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest multiplied by the molecular weight of the (now identified, such that it's amino acid sequence or sequences are available) protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. In some embodiments, the molecular weight of a protein complex is the combined molecular weight of its subunits. The molecular weight of a subproteome or proteome of interest is the mean of the molecular weights of the proteins, peptides, oligopeptides, polypeptides, and / or protein complexes which comprise the proteome or subproteome of interest. In some embodiments, the concentration of a proteome is a measure of the viral load, bacterial load and / or parasitic load of a proteome, or mixture of proteomes in the sample. In some embodiments, the proteome is a viral proteome, and the method provides the total molar protein concentration of the viral proteome within the sample. This is equivalent to the traditional viral load measurement in copies / mL. Alternatively, the method provides the viral load measurement in copies / mL using standard techniques known in the art. In some embodiments, the proteome is a bacterial proteome, and the method provides the total bacterial concentration of the bacterial proteome within the sample. This is equivalent to the bacterial load measurement in colony forming units (CFU). Alternatively, the method provides the bacterial load measurement in CFU using standard techniques known in the art. In some embodiments, the proteome is a parasitic proteome and the method provides the total parasitic concentration of the parasitic proteome within the sample. This is equivalent to the parasitic load measurement in number of parasites per host sample. Alternatively, the method provides the parasitic load measurement in number of parasites per host sample using standard techniques known in the art. Although we have described certain embodiments, in relation to molar concentration, these embodiments are equally applicable to mass concentration, as has been described in the examples.

[0092] As used herein, the term "concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest" is an abbreviation to refer to the protein concentration of the protein of interest, or, peptide concentration of the peptide of interest, or, oligopeptide concentration of the oligopeptide of interest, or, polypeptide concentration of the polypeptide of interest, or, protein complex concentration of the protein complex of interest, or, subproteome concentration of the subproteome of interest, or, proteome concentration of the proteome of interest.

[0093] As used herein, the term "amount" refers to the number of moles of entities within a sample. An entity can be a molecule, a complex, a monomer within a polymer such as an amino acid contained within a protein chain, or an atom. The number of moles of entities is the total number of entities contained within the sample divided by the Avogadro constant N A , which is 6.02214076×10 23< mol -1< . Unless otherwise stated, the amount refers to the number of moles of molecules within a sample. The amount refers to the number of moles of molecules of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes or proteomes of interest in the sample. In some embodiments, the amount of a protein complex containing multiple protein subunits considers the entire protein complex as one molecule. A proteome or subproteome of interest has many different types of molecules. The amount of a subproteome or proteome of interest refers to the total number of moles of proteins, peptides, oligopeptides, polypeptides, and protein complexes that comprise the proteome or subproteome of interest within the sample. In some embodiments, the molar concentration of the sample is multiplied by the volume of the sample to provide the amount of the sample.

[0094] As used herein, the term "relative concentration" refers to fold changes in the concentration of molecules between samples. For example, a first sample that has been diluted from a second sample has a lower relative concentration than the second sample.

[0095] As used herein, the term "amino acid concentration" refers to the molar or mass concentration of amino acids within an amino acid type. Amino acid concentration refers to the amount or mass of amino acids within an amino acid type per unit of volume. Unless otherwise stated, the term amino acid concentration refers to the molar concentration of amino acids within an amino acid type. The molar concentration of amino acids within an amino type may be different than the concentration of molecules, because more than one amino acid of an amino acid type, or zero amino acids of an amino acid type, can be contained within a molecule. The amino acid concentration of amino acids within an amino acid type is equal to the total molar concentration of molecules multiplied by the number of amino acids of the amino acid type per molecule. For example, if the molecules are proteins, then the amino acid concentration of an amino acid type can be (and is usually) different than the protein concentration. The amino acid concentration of an amino acid type within a sample is calculated from the measured value of the label of that amino acid type within the sample, using a calibration curve or standard providing the value of the label for one or more proteins or amino acids of known amino acid concentration. Importantly, the amino acid concentration of three or more amino acid types of a sample does not refer to the concentration of the sample. The amino acid concentrations of three or more amino acid types of a protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest does not refer to the concentration of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest.

[0096] As used herein, the term "protein" refers to a biomolecule or macromolecule comprised of one or more linear polypeptide chains of amino acids. A protein is a polymer of amino acids. The term "protein" includes, but is not limited to, molecules which contain from about 50 to about 3000 amino acids. The term "protein" refers to one or more polypeptide chains arranged in a way which is often biologically functional. A protein can have a 3-dimensional structure which is folded, 3-dimensional structure which is intrinsically disordered or a 3-dimensional structure which is partially folded and partially disordered. A protein also refers to a biomolecule or macromolecule comprised of one or more linear polypeptide chains of amino acids that also includes other components. For example, a protein also includes glycoproteins (in which chains of sugar molecules are covalently attached to protein molecules), or a nucleoprotein in which a protein is associated with or bonded to a nucleic acid.

[0097] As used herein, the term "peptide" refers to short chains of amino acids linked by peptide (amide) bonds. The term "peptide" includes, but is not limited to, molecules that contain from about 2 to about 50 amino acids. In a preferred aspect, the term "peptide" refers to molecules that contain greater than 10 amino acids.

[0098] As used herein, the term "oligopeptide" refers to a class within peptides that includes, but is not limited to, molecules that contain from about 2 to about 20 amino acids. The term "oligopeptide" includes, but is not limited to, dipeptides that contain 2 amino acids, tripeptides that contain 3 amino acids, tetrapeptides that contain 4 amino acids, and pentapeptides that contain 5 amino acids.

[0099] As used herein the term "polypeptide" is a single linear chain of many amino acids, held together by peptide bonds.

[0100] As used herein, the term "protein complex" refers to a structurally associated group of two or more subunits containing at least one protein subunit. A protein complex often contains two or more proteins. It can also contain one or more proteins and one or more nucleic acids (ribonucleoproteins). Protein complexes are a form of stable protein-protein interactions in which the protein subunits usually cooperate to perform a biological function. An example of a protein complex is a ribosome. Because the protein subunits within protein complexes are stably structurally associated with one another and cooperate to form a biological function, the number of amino acids of each of three or more amino acid types within each subunit of the protein complex are summed to determine the number of amino acids of each of three or more amino acid types for the protein complex.

[0101] As used herein, the term "protein-protein interaction" refers to an interaction between protein molecules, usually involving specific physical contacts. Protein-protein interactions can be stable or transient. In the methods of the invention, protein-protein interactions which do not comprise protein complexes, such as transient protein interactions, are treated as protein mixtures.

[0102] As used herein, the term "subproteome" is a collection of proteins that are part of a proteome and share a common characteristic, such as being disease-associated. For example, a subproteome within the human plasma proteome is the heart disease subproteome. A disease-associated subproteome can include all or some of the proteins within a proteome. A subproteome can also describe proteins within a proteome that share a common physical characteristic, such as, but not limited to being low molecular weight, size, charge and / or density. In some embodiments, low molecular weight characteristics refers to proteins of less than 10kDa, less than 30 kDa, less than 50kDa, less than 100kDa, 10-30 kDa, 30-50 kDa, 10-50kDA , 30-50kDA, 10-100kDa, 50-100kDa or 30-100 kDa. In preferred embodiments, low molecular weight refers to proteins of less than less than 10kDa, less than 30 kDa, less than 50kDa, less than 100kDa, or proteins of 10 kDa, 30 kDa, 50 kDa or 100 kDa. In preferred embodiments, low molecular weight refers to proteins of less than 50kDa or proteins of 50kDa. In some embodiments, charge characteristics refers to chromatography including ion-exchange chromatography that can be used to select proteins that bind to oppositely charged resins. In some embodiments, density characterisitcs refers to sedimentation coefficient which is related to protein size and shape.

[0103] As used herein, the term "proteome" refers to all of the proteins expressed by an organism. The term "proteome" also refers to all the proteins expressed by an organism within a particular tissue type, for example, the human plasma proteome. The term "proteome" also refers to all the proteins expressed within a particular cell type, for example, glioblastoma cells. The term "proteome" also refers to changes in the proteins expressed by an organism, tissue type, or cell type at a given time or under a given set of conditions, for example when treated with a drug. The term "proteome" includes, but is not limited to, viral proteomes, bacterial proteomes, archaea proteomes, parasitic proteomes, yeast proteomes, plant proteomes, animal proteomes, mammalian proteomes, and the human proteome. The term "proteome" includes, but is not limited to, viral proteomes with less than 50 proteins, bacterial proteomes with less than 7000 proteins, the human plasma proteome with less than 5000 proteins, the human urine proteome with less than 5000 proteins, the human salivary proteome with less than 5000 proteins, and the human proteome with approximately 22000 proteins.

[0104] As used herein, the term "mixture" refers to two or more proteins, peptides, polypeptides or oligopeptides, subproteomes and / or proteomes in a sample. For example, a mixture of peptides is a combination of two or more peptides, a mixture of polypeptides is a combination of two or more polypeptides, and a mixture of proteins is a combination of two or more proteins. The mixture does not have to be comprised of the same components. For example, a mixture can also be a mixture of proteins and peptides, a mixture of peptides and polypeptides, a mixture of proteins and polypeptides etc.

[0105] As used herein, the "sample" refers to any sample that may contain one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest. The term "sample" also includes any sample that does not contain any proteins and thus no value (e.g. signal of the label) is obtained when the label is measured.

[0106] As used herein, the term "amino acid type" refers to organic compounds that comprise one amine (-NH) and one carboxyl (-CO) group, one alpha carbon, and one R group (side chain) specific to each amino acid type, or that comprise one amine (-NH 2 ) and one carboxyl (-COOH) group, one alpha carbon, and one R group (side chain) specific to each amino acid type, or that comprise one amine (-NH 2 ) and one carboxyl (-CO) group, one alpha carbon, and one along R group (side chain) specific to each amino acid type, or that comprise one amine (-NH) and one carboxyl (-COOH) group, one alpha carbon, and one R group (side chain) specific to each amino acid type, or, describing the amino acid type proline, amino acid type also refers to organic compounds that comprise one imine (-NH) and one carboxyl (-COOH) group, one alpha carbon, and one R group (side chain) specific to each amino acid type, or that comprise one imine (-NH) and one carboxyl (-CO) group, one alpha carbon, and one R group (side chain) specific to each amino acid type. Amino acid type includes both free amino acids and amino acids within protein sequences. Amino acids within protein sequences can alternatively be called amino acid residues or residues. The amino acid type is defined by the R-group (side chain) specific to each amino acid type. The term amino acid type refers to a proteinogenic amino acid selected from: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), pyrrolysine (O), proline (P), selenocysteine (U), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V), or, a non-proteinogenic synthetic amino acid, including, but not limited to non-proteinogenic synthetic amino acids that contains the functional groups azide, alkyne, alkene, diene, acyl, iodo and / or boronic acid. The term "amino acid type" encompasses modified amino acids, unmodified amino acids and / or a combination of both modified and unmodified amino acids of an amino acid type. In some embodiments, the term "amino acid type" refers to modified amino acids of an amino acid type. In some embodiments, the term "amino acid type" refers to unmodified amino acids of an amino acid type. In some embodiments, the term "amino acid type" refers to a combination of modified and unmodified amino acids of an amino acid type. In some embodiments, the term "amino acid type" refers to both the unmodified amino acids of an amino acid type and the combination of the modified and the unmodified amino acids of an amino acid type.

[0107] As used herein, the term "R-group" refers to the side chain present in each amino acid of each amino acid type. The R-group is a substituent; an atom, or group of atoms which replaces one or more hydrogen atoms on the alpha carbon of the amino acid. The R-group of each amino acid type is unique for that amino acid type. The R-groups of each amino acid type encompassed by the invention are defined in Table 2. An amino acid type is defined by the R-group present on the unmodified (as translated) amino acid type. If subsequent modifications are made to the R-group, the amino acid type does not change. For example, the cysteine (C) amino acid type is defined by the thiol R-group. This is the R-group of unmodified amino acids of the cysteine amino acid type (reduced cysteine, C R ). A subset of cysteine amino acids within the cysteine amino acid type can be post-translationally modified to form cysteine disulphide (C D ), and this same subset of cysteine amino acids can be reduced to form reduced cysteine (C R ). The amino acid type remains cysteine (C) during these transformations. This is the case regardless of whether a post-translational modification or other modification is reversible or irreversible.

[0108] As used herein, a "modified amino acid" refers to amino acids of an amino acid type that have been chemically modified after being incorporated into a protein. In some embodiments, an enzyme carries out this chemical modification. In some embodiments, the modified amino acids have undergone post-translational modification. Examples of such post-translational modification of amino acids include, but are not limited to, methylation, deamination, deamidation, N-linked glycosylation, isomerization, disulfide-bond formation, oxidation to sulfenic, sulfinic or sulfonic acid, palmitoylation, N-acetylation (N-terminus), S-nitrosylation, cyclization to pyroglutamic acid (N-terminus), gamma-carboxylation, isopeptide bond formation, N-Myristoylation (N-terminus), phosphorylation, acetylation, ubiquitination, SUMOylation, methylation, hydroxylation, oxidation to sulfoxide or sulfone, hydroxylation, O-linked glycosylation, mono- or di-oxidation, formation of Kynurenine, and / or sulfation. For example, the amino acids of the amino acid type cysteine (C) can be modified during post-translational modification to form cysteine disulphide (C D ) amino acids that contains disulphide bonds and an oxidized thiol R-group.

[0109] As used herein, an "unmodified amino acid" refers to amino acids of an amino acid type that have not been chemically modified after being incorporated into a protein. For example, the unmodified amino acids of the amino acid type cysteine (C) are reduced cysteine (C R ); reduced cysteine (C R ) is not disulphide bonded and has not undergone any other post-translational modification, and contains a reduced thiol.

[0110] As used herein, the term "three or more amino acid types" refers to at least three amino acid types. The term "three or more amino acid types" encompasses, but is not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid types. In some embodiments, 3 amino acid types are labelled. In some embodiments, 4 amino acid types are labelled. In some embodiments, 5 amino acid types are labelled. In some embodiments, 6 amino acid types are labelled. In some embodiments, 7 amino acid types are labelled. In some embodiments, 8 amino acid types are labelled. In some embodiments, 9 amino acid types are labelled. In some embodiments, 10 amino acid types are labelled. In some embodiments, 11 amino acid types are labelled. In some embodiments, 12 amino acid types are labelled. In some embodiments, 13 amino acid types are labelled. In some embodiments, 14 amino acid types are labelled. In some embodiments, 15 amino acid types are labelled. In some embodiments, 16 amino acid types are labelled. In some embodiments, 17 amino acid types are labelled. In some embodiments, 18 amino acid types are labelled. In some embodiments, 19 amino acid types are labelled. In some embodiments, 20 amino acid types are labelled. In some embodiments, 21 amino acid types are labelled. In some embodiments, 22 amino acid types are labelled. In some embodiments, 23 amino acid types are labelled. In some embodiments, 24 amino acid types are labelled. In some embodiments, 25 amino acid types are labelled. In some embodiments, 26 amino acid types are labelled. In some embodiments, 27 amino acid types are labelled. In some embodiments, 28 amino acid types are labelled. In some embodiments, 29 amino acid types are labelled. In some embodiments, 30 amino acid types are labelled. In some embodiments, 31 amino acid types are labelled. In some embodiments, 32 amino acid types are labelled. In some embodiments, 33 amino acid types are labelled. In some embodiments, 34 amino acid types are labelled. In some embodiments, 35 amino acid types are labelled. In some embodiments, 36 amino acid types are labelled. In some embodiments, 37 amino acid types are labelled. In some embodiments, 38 amino acid types are labelled. In some embodiments, 39 amino acid types are labelled. In some embodiments, 40 amino acid types are labelled.

[0111] As used herein, the term "label" or "labelled" refers to a tag, identifier, or probe that is added, inserted, attached, bound, or bonded to the amino acids within an amino acid type to aid the detection and / or identification of the amino acid type within the sample. For example, a label can include a fluorophore, an isotope, or a tandem mass tag. In some embodiments, the label provides a signal. In some embodiments, the label is a fluorescent label. In some embodiments, the label is a fluorogenic dye, or a molecule which becomes fluorescent upon reaction with an amino acid type. In some embodiments, the label is covalently bonded to the amino acids within an amino acid type. In some embodiments, the label is covalently bonded to the R-group of amino acids within an amino acid type.

[0112] As used herein, the term "signal" refers to an occurrence that conveys information. In some embodiments, a signal is a time-varying occurrence that conveys information. The signal of a label can be read at a single point in time, or the signal of a label can be read as a function of time. In some embodiments, the label is a fluorescent label and the signal of the label is fluorescence intensity.

[0113] As used herein, the term "luminescence" refers to spontaneous emission of light by a substance not resulting from heat. In some embodiments, label is a luminescent label and the signal of the label is a luminescent signal. There are several types of luminescence, including but not limited to photoluminescence (which includes fluorescence), chemiluminescence (which includes bioluminescence), electroluminescence, radioluminescence, and thermoluminescence. Photoluminescence is the result of absorption of photons. There are several types of photoluminescence, including fluorescence which is photoluminescence as a result of singlet-singlet electronic relaxation with a typical lifetime of nanoseconds. Phosphorescence is another type of photoluminescence which is the result of triplet-singlet electronic relaxation with a typical lifetime of milliseconds to hours. Chemiluminescence is the emission of light as a result of a chemical reaction. Bioluminescence is a form of chemiluminescence which is the result of biochemical reactions in a living organism. Electrochemiluminescence is the result of an electrochemical reaction. Electroluminescence is a result of an electric current passed through a substance. Cathodoluminescence is the result of a luminescent material being struck by electrons. Sonoluminescence is the result of imploding bubbles in a liquid when excited by sound. Radioluminescence is the result of bombardment by ionizing radiation. Thermoluminescence is the re-emission of absorbed energy when a substance is heated. Cryoluminescence is the emission of light when an object is cooled.

[0114] As used herein, the term "calibration curve" or the term "standard" refers to a general analytical chemistry method for determining the concentration of a substance in an unknown sample by comparing the unknown sample to a set of standard samples, or one standard sample, of known concentration. If the unknown sample is compared to a set of standard samples, "calibration curve" is used. If the unknown sample is compared to a single standard sample, the term "standard" is used. A calibration curve or standard is used to convert between known amino acid concentration and measured label (e.g. signal of the label) of each of three or more amino acid types for the protein of interest, or, to convert between measured label (e.g. signal of the label) of the same three or more amino acid types and the amino acid concentration of each amino acid type in the sample. A calibration curve for an amino acid type refers to data (signal of the label) collected for several known amino acid concentrations of the amino acid type, and a standard refers to data (signal of the label) collected for one known amino acid concentration of the amino acid type. A calibration function or (scalar) calibration factor is calculated from the calibration curve or standard.

[0115] As used herein, the term "proportion" refers to any number of amino acids of an amino acid type that is less than all of the amino acids of an amino acid type in the sample, i.e. less than 100% of the amino acids of an amino acid type in the sample. The term "proportion" also refers to any number of amino acids of an amino acid type that is less than all of the subset of the amino acids of the amino acid type that react with the label (e.g. unmodified amino acids of an amino acid type), for example according to the rules provided in Table 4. The term "proportion" includes, but is not limited to, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% of amino acids of each amino acid type being labelled in the sample. In some embodiments, a proportion is about 50% of the amino acids of a particular amino acid type present in the sample. In some embodiments, a proportion is about 60% of the amino acids of a particular amino acid type present in the sample. In some embodiments, a proportion is about 70% of the amino acids of a particular amino acid type present in the sample. In some embodiments, a proportion is about 80% of the amino acids of a particular amino acid type present in the sample. In some embodiments, a proportion is about 90% of the amino acids of a particular amino acid type present in the sample.

[0116] As used herein, the term "measuring" refers to the detection and quantification. In some embodiments, measuring includes measuring a signal.

[0117] As used herein, the term "number of amino acids" refers to the number of amino acids of a certain amino acid type per molecule. To determine the number of amino acids of each labelled type in a sample, the amino acid concentration of an amino acid type in a sample is divided by the molar protein concentration of the sample. To determine the number of amino acids of an amino acid type in a protein of interest, or reference, the number of amino acids of an amino acid type is calculated from the protein sequence of the protein of interest, or, has been previously determined and for example is accessible via a database. Alternatively, the number of amino acids of an amino acid type in a protein of interest can be determined by labelling an amino acid type in the protein of interest at a known protein concentration, measuring the label, converting the measuring label to the amino acid concentration using the methods disclosed herein and dividing the amino acid concentration of the amino acid type by the molar protein concentration of the protein of interest. For example, if lysine is the amino acid type being labelled and there are 54 lysine's per protein molecule in the sample, then the number of amino acids of the amino acid type of lysine is 54. The number of amino acids of an amino acid type does not refer to the total number of amino acids of an amino acid type in a solution containing the sample. For example, if there are 10000 protein molecules in the sample, and each protein molecule contains 54 lysine amino acids, then the number of amino acids of the lysine amino acid type is 54, not 540000.

[0118] As used herein, the term "background correct" or "background corrected" refers to the measured label of each labelled amino acid type which has been corrected to exclude any signal from the free label in solution not added, inserted, attached, bound, bonded or covalently bonded to amino acids of the amino acid type of interest, non-specific labelling, or other sources of signal that would otherwise contribute to the total label being measured, such as cellular autofluorescence. This is achieved by standard means in the art.

[0119] As used herein, the term "bulk" refers to studies performed without constraining the sample within channels that have dimensions of in general hundreds of micrometers or less. Classically, bulk studies do not involve manipulation of small amounts (picoliters to nanoliters) of fluids, and fluids mix turbulently in addition to diffusively. Bulk studies include the automated manipulation of fluids, for example by pumps or robots. Bulk studies can involve analysing samples in plates, which have sample reservoirs to perform many reactions and / or measurements in parallel, and can involve using a plate reader or similar instrument. Generally, bulk studies do not seek to detect single protein molecules.

[0120] As used herein, the term "solution phase" refers to studies performed and measured in solution. Solution phase excludes methods which require measurement on a surface, such as transforming internal reflection fluorescence (TIRF) microscopy. Solution phase excludes methods that require proteins within a sample to be passed through synthetic or natural pores within a surface. For example, solution phase excludes methods incorporating nanopores, small channels within surfaces, and excludes methods incorporating biological nanopores, transmembrane proteins embedded within lipid membranes.

[0121] As used herein, the term "deconvolute" refers to a process in which a signal deriving from multiple components is analyzed or transformed to reveal the portions from each component. In some embodiments, if a time-resolved signal derives from two components and there are two separated peaks, then a signal can be deconvoluted kinetically such that analysis of one peak provides information about one component and analysis of the other peak provides information about the other component. For example, kinetic deconvolution can be used if the label is a fluorescent label and two or more amino acid types are labelled with the same fluorescent label under the same conditions, but the labelling reactions proceed at different rates, such that measuring the signal of the label at a certain time provides information about exclusively one amino acid type, and measuring the signal of the label at another time provides information about exclusively another amino acid type. Alternatively, if a signal derives from two components and one component is known, the signal can be transformed to remove the known component and only reveal information about the unknown component.

[0122] As used herein, the term "deconvolution standard" refers to a protein of known amino acid concentration of the three or more amino acid types labelled and measured in the sample which is used to deconvolute the signals obtained when three amino acid types are labelled with the same label under the same conditions. A deconvolution standard can be measured at different excitation and emission wavelengths, to deconvolute the contribution of each labelled amino acid type at each wavelength and enable separation of the signals of each labelled amino acid type in the sample. A deconvolution standard is not a "calibration curve or standard" discussed above.

[0123] As used herein, the term "protein sequencing" refers to determining the sequence of amino acids in a protein, peptide, oligopeptide, or polypeptide. Protein sequencing involves consecutively reading and identifying single amino acids along an amino acid sequence, starting at one terminus of the amino acid chain, and moving, one amino acid at a time, along the amino acid chain. Protein sequencing determines the positions of amino acids within a protein. For example, Edman degradation is a common method of protein sequencing.

[0124] As used herein, the term "n-dimensional space" refers to a mathematical space in which n is the minimum number of coordinates needed to specify any point within it. Within an n-dimensional space, there are n-dimensions of information. The dimensions of information are the number of amino acid types being labelled. For example, 3 dimensions of information refers to 3 amino acid types being labelled and requires a 3-dimensional space. In the present invention, an n-dimensional space is used to plot the values of the label, amino acid concentrations, or number of amino acids for n amino acid types. In an n-dimensional space, there are n coordinates necessary to specify any vector.

[0125] As used herein, the term "reference" is a standard or control value against which the value of the sample is compared. The reference can include information indicating the known label values, and / or amino acid concentrations, and / or number of amino acids of the same three or more amino acid types as the amino acid types that have been labelled in the sample for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. The reference can include the known label values (e.g. signal, e.g. fluorescence intensity) or the amino acid concentrations of three or more amino acid types of the protein, peptide, oligopeptide, polypeptide, protein complex subproteome or proteome of interest at one or more protein concentrations, or, the number of amino acids of three or more amino acid types in the amino acid sequence or sequences of the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome or proteome of interest. The three or more amino acid types are the same three or more amino acid types that have been labelled in the sample. The reference for each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest is used to identify the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest within a sample. In some embodiments, the reference is the weighted mean of the known label values, amino acid concentration or number of amino acids of three or more amino acid types across all of the amino acid sequences of a proteome or subproteome, weighted by the proportion of each protein across the proteome, subproteome or mixture of proteins. In some embodiments, the reference is stored in, and accessed / obtained from, a database. In some embodiments, the reference is experimentally determined. In some embodiments, the reference is calculated from the amino acid sequence or sequences of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes or proteomes of interest. In some embodiments, creating the reference includes accessing the publicly available amino acid sequences of a variety of proteins and removing the portions of the sequence that are biologically cleaved in the mature proteins. In some embodiments, creating the reference includes determining the number of amino acids of the same three or more amino acid types as have been labelled in the sample within the amino acid sequence or amino acid sequences of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest, having optionally applied the rules outlined in Table 4 to remove from the number of amino acids of an amino acid type post-translationally modified amino acids that would not react with the label for the amino acid type. In some embodiments, the reference is determined using the methods disclosed herein, i.e. labelling three or more amino acid types, measuring the label and using the measured label to determine the number of amino acids of each amino acid type in the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest or the concentration of amino acids of each amino acid type in a sample containing each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. In some embodiments, the reference provides the known label values and / or amino acid concentrations of the same three or more amino acid types as the amino acid types that have been labelled in the sample of each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest as a set of parametric equations or a vector function depending on the common parameter of concentration. In other embodiments, the reference provides the number of amino acids of the same three or more amino acid types as the amino acid types that have been labelled in the sample of each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest. In some embodiments, the reference includes concentration ranges for the protein, peptide, oligopeptide, polypeptide, protein complex, subproteome or proteome of interest, that are known or that are determined using the methods of the invention. In some embodiments, these known concentration ranges are used as bounds on the function or functions which comprise the reference. In some embodiments, the reference includes additional information, such as information incorporating observed experimental error rates. In some embodiments, the reference includes information derived from Benford's law which provides the frequency distribution of leading digits within many datasets observed in nature.

[0126] As used herein the term "single reference" refers to a reference provided for a proteome and / or subproteome of interest uniquely identifying the proteome and / or subproteome of interest on the basis of its average composition. Although many individual proteins may be contained in a proteome and / or subproteome of interest, it is not necessary to provide the known label values, amino acid concentrations and / or number of amino acids as a reference for each protein contained within the proteome and / or subproteome of interest in order to identify the proteome and / or subproteome of interest. For example, if a proteome of interest contains 700 proteins, it is not necessary to provide a the known label values, amino acid concentrations and / or number of amino acids as a reference for all 700 proteins contained within the proteome and / or subproteome of interest. Instead, the single reference provided for the proteome and / or subproteome of interest provides the average signature of the proteome and / or subproteome of interest, permitting its identification. For example, the single reference for the colorectal cancer proteome of interest in blood plasma permits the identification of the colorectal cancer proteome of interest from blood plasma via only labeling and measuring three or more amino acid types within the blood plasma solution and comparing the measured values of the label or amino acid concentrations calculated from the measured values of the label to the values provided by the single reference. There is no need to measure individual proteins and / or biomarkers within the colorectal cancer proteome and / or subproteome of interest in order to detect the presence and / or concentration and / or amount of the colorectal cancer proteome and / or subproteome of interest. A proteome and / or subproteome of interest is identified and it's concentration / amount determined without any requirement to measure a single protein within it. The single reference for a proteome and / or subproteome of interest can be calculated theoretically or experimentally using the methods of the invention and is an algebraic function of the total protein concentration of the proteome and / or subproteome of interest, which can for example be described by one of the vector functions or sets of parametric equations described herein.

[0127] As used herein, the term "reduced cysteine" (C R ) refers to unmodified amino acids of the amino acid type cysteine (C), which have a reduced thiol R-group. Reduced cysteine is unmodified because it is not disulphide bonded during post-translational modification and has not undergone any other post-translational modification of the thiol R-group such as oxidation to sulfenic, sulfinic or sulfonic acid, palmitoylation, or S-nitrosylation. The term "reduced cysteine" (C R ) is equivalent to the term "free cysteine" known in the art.

[0128] As used herein, the term "cysteine disulphide" (C D ) refers to modified amino acids of the amino acid type cysteine (C), in which a thiol R-group has undergone an oxidative coupling reaction with another thiol R-group resulting in the formation of a disulphide bond. Cysteine disulphide (C D ) has an oxidized thiol R-group. Cysteine disulphide (C D ) is a type of reversible post-translational modification of the amino acid type cysteine (C). The number of cysteine disulphides refers to the number of cysteine amino acids engaged in disulphide bonds, not the number of disulphide bonds which is ½ the number of cysteine disulphides engaged in disulphide bonds because one disulphide bond comprises two cysteine amino acids.

[0129] As used herein, the term "cysteine" (C) refers to unmodified amino acids of cysteine (C R ), modified amino acids of cysteine (C D ) and / or the combination of unmodified and modified amino acids of cysteine. In order to label both modified and unmodified cysteine amino acids , first the cysteine disulphide (C D ) amino acids are chemically reduced to reduced cysteine (C R ), such that they are available for reaction with the label.

[0130] As used herein, the term "classifier" refers to an algorithm that implements classification. Classification is the identification of a category to which a new observation belongs, on the basis of a training set of data that contains observations whose category membership is known. The term "classifier" encompasses a machine learning classifier that uses supervised learning to learn a function that maps an input to an output based on example input-output pairs, including using both lazy learning (instance-based learning) and eager learning. For example, a classifier describes a k-nearest neighbor classifier (lazy learning) and / or a support vector machine classifier (eager learning). The classifier can be used in the comparison step of the methods described herein.

[0131] As used herein, the term "duplicate" refers to rare a case in which more than one protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest have the same reference, or where the references for more than one protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest are indistinguishable based on a comparison of values of the label of three or more amino acid types, amino acid concentration, or number of amino acids of three or more amino acid types. This occurs because the number of amino acids of three or more amino acid types in one protein of interest is the same as, or a multiple of, the number of amino acids of the same three or more amino acid types in another protein of interest. A reference can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 duplicates, but more than 1 duplicate is rare. If two proteins of interest have the same reference, and this reference is identified within the sample, then the sample is identified as containing either of these two proteins of interest. There are a number of approaches to eliminate this effect and uniquely identify the protein of interest present within the sample, such as comparing the protein concentration at which the reference has been identified in the sample to the known biologically relevant concentration ranges of both proteins of interest. The sample is identified as containing the protein of interest which is within its biologically relevant concentration range.

[0132] For the sake of clarity and explanation, the methods are described in the context of a protein or proteome of interest. However, unless otherwise specified or made clear from the context, the methods of the invention should be understood to be generally, additionally, or alternatively, applicable to one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest.Sample

[0133] The samples utilised in the present methods have been obtained from the subject using standard methodology. Preferably, the sample is a bodily fluid sample, tissue sample, soil sample, water sample, environmental sample, crop sample, food sample, drink sample or laboratory sample.

[0134] Bodily fluid samples encompassed by the invention include, but are not limited to: whole blood samples, blood serum samples, blood plasma samples, salvia samples, sputum samples, faeces samples, urine samples, semen samples, nasal swab samples, nasopharyngeal aspirate samples, throat swab, or lower respiratory samples, such as a lower respiratory mucus aspirate sample, cerebrospinal (CSF) sample, sexual health sample, such as a urethral swab, cervix swab, vaginal swab or rectal swab. Alternatively, the sample can contain any other bodily fluid known in the art. In some embodiments, the bodily fluid sample is any type of fluid produced by a lesion. In some embodiments, the sample is a blood plasma sample. In some embodiments, the sample is a platelet poor plasma (PPP) sample. In some embodiments, the sample is a platelet rich plasma (PPP) sample. In some embodiments, the sample is a platelet sample. In some embodiments, the sample is a blood plasma exosome sample. In some embodiments, the sample is a blood cell sample. In some embodiments, the blood cell sample is a lymphocyte sample or a myeloid cell sample. In some embodiments, the sample is a urine sample.

[0135] Alternatively, the sample may be a tissue sample. Preferably, the tissue sample is a biopsy of any tissue type of interest. For example, the tissue sample can be a biopsy of a solid tumor. This includes, for example, sarcomas, lymphomas, carcinomas and melanomas.

[0136] Alternatively, the sample may be an environmental sample. Preferably, the environmental sample is a water sample, such as a drinking water sample or wastewater sample. In some embodiments, the sample is a sample suspected of biological warfare.

[0137] Alternatively, the sample may be a food sample, for example in the food industry. For example, the methods of the invention may be used to test a food sample for bacterial growth and composition, for example in cheese making, testing for flour and bread quality in bread making such as via assessing the strength of gluten, quantifying the amount of a fermentation agent (for example, identifying and quantifying the amount of bacteria in kombucha to ensure it is safe to consume), testing yoghurt, or testing a sourdough mother culture. Preferably, the food sample is suspected of containing an allergen.

[0138] In some embodiments, the sample can be suspected of containing an allergen. Preferably, the allergen is peanuts, gluten, lactose, pollen or dust mites, dust, caseins, lipocalins, c-type lysozymes, protease inhibitors, tropomyosins, parvalbumins, cat dander, dog dander.

[0139] Alternatively, the sample may be a drink sample such as a milk sample, a water sample or a fruit juice sample. For example, the methods of the invention could be used in the agriculture industry to measure a chemical signature of the hormone component of milk, or to assess unpasteurized milk or fruit juices for bacterial contamination.

[0140] In some embodiments, the sample is a bodily fluid sample (e.g. whole blood samples, blood serum samples, blood plasma samples, salvia samples, sputum samples, faeces samples, urine samples, semen samples, nasal swab samples, nasopharyngeal aspirate samples, throat swab, or lower respiratory samples, such as a lower respiratory mucus aspirate sample, cerebrospinal (CSF) sample, sexual health sample, such as a urethral swab, cervix swab, vaginal swab or rectal swab, or any type of fluid produced by a lesion), a tissue sample, a soil sample, an environmental sample (e.g. water sample such as a drinking water sample or wastewater sample; or sample suspected of biological warfare), a food sample (e.g. suspected of containing an allergen such as peanuts, gluten, lactose or pollen, caseins, lipocalins, c-type lysozymes, protease inhibitors, tropomyosins, parvalbumins, cat dander and / or dog dander, or a functional foods sample) or drink sample (e.g. milk, water, fruit juice).

[0141] In some embodiments, the proteins are isolated from the sample using standard techniques in the art such as centrifugation, filtration, extraction, precipitation and differentiation solubilization, ultracentrifugation, size exclusion chromatography, separation based on charge or hydrophobicity (examples include hydrophobic interaction chromatography, ion exchange chromatography, and / or free-flow electrophoresis), and / or affinity chromatography such as immunoaffinity chromatography or high-performance liquid chromatography (HPLC). The proteins within the sample can also be concentrated once isolated. This can involve, but is not limited to, lyophilization or ultrafiltration. For example, if the sample is a saliva sample, and the presence of viruses or bacteria are being detected in the sample, the viral and bacterial proteins in the sample are separated from the human protein in the sample by centrifugation. After centrifugation, the pellet corresponds to the viruses and bacteria present in the sample, without the human proteins present within the supernatant. In another example, if the sample is a solid tissue sample, and the presence of viruses or bacteria are being detected in the sample, the viral and bacterial proteins in the sample are separated from the human protein in the sample by freezing the tissue sample, crushing the sample and extracting the protein from the tissue into a buffer. An example of these technique, which is standard in the art for extracting proteins from tissue samples is provided by January Ericsson, C. Protein Extraction from Solid Tissue. 2011. Methods in molecular biology (Clifton, N.J.) 675:307-12. DOI: 10.1007 / 978-1-59745-423-0_17.

[0142] The sample may be suspected of containing the presence of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, or proteomes of interest. In some embodiments, the proteins, peptides, oligopeptides, polypeptides, protein complexes, or proteomes of interest are isolated from other proteins in the sample.Protein of interest

[0143] Although a "protein of interest" is referred to throughout this application, the term "protein of interest" is provided as an example and can be substituted with peptide of interest, oligopeptide of interest, polypeptide of interest, proteome complex of interest, subproteome of interest, or proteome of interest, or combination thereof, whose presence and / or concentration and / or amount within the sample is being tested. In this general sense of the term, "protein of interest" is suspected of being in the sample, and the hypothesis of the protein of interest being within the sample is tested via the methods of the invention.

[0144] In some embodiments, the proteome of interest is a viral proteome, bacterial proteome, fungal proteome or parasitic proteome that is suspected of causing a viral infection, bacterial infection, fungal infection or parasitic infection, respectively. For example, in some embodiments, the subject is suspected of having malaria and the proteomes of interest include P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi proteomes. These parasites are the known causative agents of malaria. A sample, such as a blood sample is obtained from a subject suspected of having malaria, and the parasitic proteomes are separated from the blood using filtration. The parasitic proteins isolated from the blood sample are tested for the presence of any one of P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi proteomes in order to confirm the diagnosis of Malaria and identify the particular parasite causing Malaria in the subject's sample.

[0145] In some embodiments, the proteome of interest is a viral proteome. For example, in some embodiments, a subject is showing symptoms of a dry cough, tiredness, muscle aches and fever and so the subject is suspected of having influenza or coronavirus. A sample, such as a blood sample, nasal swab, nasopharyngeal aspirate or lower respiratory mucus aspirate sample is obtained from the subject and the sample is tested for the presence of Influenza proteomes, for example the Influenza A H1N1 proteome, and / or Coronavirus proteomes, for example the SARS-CoV-2 (Covid-19) proteome to identify the virus causing the symptoms in the subject and thus identify the infection that the subject has.

[0146] In some embodiments, the proteome of interest is the human proteome. In some embodiments, the proteome of interest is the human plasma proteome. In some embodiments, the albumin fraction of the human plasma proteome is removed prior to the remaining steps of the method. In some embodiments, the albumin and globulin fraction of the human plasma proteome is removed prior to the remaining steps of the method. In alternative embodiments, the albumin fraction of the human plasma proteome is not removed prior to the remaining steps of the method. In some embodiments, the albumin and globulin fraction of the human plasma proteome is not removed prior to the remaining steps of the method. In some embodiments, the albumin and globulin fraction of the human plasma proteome is removed prior to the remaining steps of the method using a centrifugal filtration step that removes high molecular weight proteins such as albumin and globulin prior to the remaining steps of the method. In some embodiments, the proteome of interest is one or more of the following human proteomes of specific glands / tissues: human eye proteome, retina, heart, skeletal muscle, smooth muscle, adrenal gland, parathyroid gland, thyroid gland, pituitary gland, lung, bone marrow, lymphoid tissue, liver, gallbladder, testis, epididymis, prostate, seminal vesicle, ductus deferens, adipose tissue, brain, salivary gland, esophagus, tongue, stomach, intestine, pancreas, kidney, urinary bladder, breast, vagina, cervix, endometrium, fallopian tube, ovary, placenta, skin, blood, or any combination thereof. The proteome of interest can also include the human metabolic proteome and / or the human secretory proteome.

[0147] In some embodiments, the proteome of interest can be a subproteome. For example, one or more human cancer subproteomes, selected from: the human pancreatic cancer subproteome, human glioma subproteome, human head and neck cancer subproteome, human thyroid gland cancer subproteome, human lung cancer subproteome, human liver cancer subproteome, human testisticular cancer subproteome, human prostate cancer subproteome, human stomach cancer subproteome, human colon / rectal cancer subproteome, human breast cancer subproteome, human endometrial cancer subproteome, human ovarian cancer subproteome, human cervical cancer subproteome, human kidney cancer subproteome, human urinary and bladder cancer subproteome, human melanoma subproteome and any combinations thereof. The following subproteomes can also be of interest: the human type I diabetes mellites subproteome, the human type II mellites diabetes subproteome, Alzheimer's disease subproteome, human Parkinson's disease subproteome, human dementia subproteome, human cardiovascular disease subproteome, human down syndrome subproteome, human aging subproteome or any combination thereof.

[0148] In some embodiments, a disease-associated sub proteome includes those proteins of an organism affected by the disease state of that organism. In some embodiments, the subproteome of interest is the human pancreatic cancer subproteome of the human blood plasma proteome. In some embodiments, the subproteome of interest is human pancreatic cancer subproteome of the human platelet poor plasma (PPP) proteome. In some embodiments, the subproteome of interest is human pancreatic cancer subproteome of the human platelet rich plasma (PRP) proteome. In some embodiments, the subproteome of interest is the human pancreatic cancer subproteome of the human blood plasma proteome. In some embodiments, the subproteome of interest is human pancreatic cancer subproteome of the human platelet poor plasma (PPP) proteome. In some embodiments, the subproteome of interest is human pancreatic cancer subproteome of the human platelet rich plasma (PRP) proteome. In some embodiments, the subproteome of interest is human prostate cancer subproteome. In some embodiments, the subproteome of interest is human colorectal cancer subproteome. In some embodiments, the subproteome of interest is human pancreatic cancer subproteome.

[0149] In some embodiments, the proteome of interest is a viral proteome. In some embodiments, the viral proteome is selected from: human papilloma virus (HPV) proteome, human immunodeficiency virus (HIV) proteome, Orthomyxoviridae proteome, Epstein Barr proteome, Ebolavirus proteome, Rabies lyssavirus proteome, Coronovirus proteome, Novovirus proteome, Hepatitis A proteome, Hepatitis B proteome, Hepatitis C proteome, Hepatitis E proteome, Hepatitis delta proteome, Herpesvirus proteome, Papillomavirus proteome, rhinovirus proteome, Measles virus proteome, Mumps virus proteome, Poliovirus proteome, rabies proteome, rotavirus proteome, west nile virus proteome, yellow fever virus proteome, Zika virus proteome, Caudovirales proteome, Nimaviridae proteome, Riboviria proteome, Inoviridae proteome, Fuselloviridae proteome, Herpesvirales proteome, Asfarviridae proteome, Bicaudaviridae proteome, tuberculosis proteome, bovine tuberculosis proteome, and any combination thereof.

[0150] In some embodiments, the Orthomyxoviridae proteome is an influenza proteome. The influenza proteome includes, but is not limited to: the Influenza A proteome, the Influenza A subtype H1N1 proteome, Influenza B proteome, Influenza C proteome or Influenza D proteome, or any combination thereof. In some embodiments, the Coronovirus proteome is the SARS-CoV-2 (Covid-19) proteome, the SARS-CoV proteome, or the MERS-CoV proteome. In some embodiments, the viral proteome of interest is a zoonotic virus proteome.

[0151] In some embodiments, the proteome of interest is a bacterial proteome. In some embodiments, the bacterial proteome includes, but is not limited to, the Escherichia coli (E. coli) proteome, Pseudomonas aeruginosa (P. aeruginosa) proteome, Salmonella proteome, Staphylococcus aureus proteome, Acinetobacter baumannii proteome, Bacteroides fragilis proteome, Burkholderia cepacia proteome, Clostridium difficile proteome, Clostridium sordellii proteome, Enterobacteriaceae proteome, Enterococcus faecalis proteome, Klebsiella pneumoniae proteome, Methicillin-resistant Staphylococcus aureus proteome, Morganella morganii proteome, Mycobacterium proteome or any combination thereof. In some embodiments, the Mycobacterium proteome is the Mycobacterium tuberculosis proteome.

[0152] In some embodiments, the proteome of interest is a parasitic proteome. In some embodiments, the parasitic proteome is selected from: a Plasmodium proteome, Toxoplasma gondii proteome, Trichomonas vaginalis proteome, Giardia duodenalis proteome, Cryptosporidiu proteome or any combination thereof. In some embodiments, the Plasmodium proteome is the Plasmodium falciparum proteome, Plasmodium knowlesi proteome, Plasmodium malariae proteome, Plasmodium ovale proteome or Plasmodium vivax proteome.

[0153] In some embodiments, the protein of interest is an allergen. Preferably, the allergen is peanuts, gluten, lactose, pollen, caseins, lipocalins, c-type lysozymes, protease inhibitors, tropomyosins, parvalbumins, cat dander and / or dog dander.

[0154] In some embodiments, the coumpound of interest is one or more proteins or peptides (e.g. alpha synuclein, lysozyme, bovine serum albumin, ovalbumin, β-Lactoglobulin, insulin, glucagon, amyloid beta, angiotensin-converting enzyme 2, angiotensin-converting enzyme, bradykinin, chordin-like protein 1, tumor necrosis factor beta, osteomodulin precursor, a matrix metalloproteinase protein, pleiotrophin, secretogranin-3, human growth hormone, insulin-like growth factor 1, leptin, telomerase, thyroid-stimulating hormone), human proteome (e.g. human plasma proteome, human eye proteome, retina, heart, skeletal muscle, smooth muscle, adrenal gland, parathyroid gland, thyroid gland, pituitary gland, lung, bone marrow, lymphoid tissue, liver, gallbladder, testis, epididymis, prostate, seminal vesicle, ductus deferens, adipose tissue, brain, salivary gland, esophagus, tongue, stomach, intestine, pancreas, kidney, urinary bladder, breast, vagina, cervix, endometrium, fallopian tube, ovary, placenta, skin, blood, human metabolic proteome, human secretory proteome), human subproteome (e.g. human cancer subproteome, selected from: the human pancreatic cancer proteome, human glioma subproteome, human head and neck cancer subproteome, human thyroid gland cancer subproteome, human lung cancer subproteome, human liver cancer subproteome, human testisticular cancer subproteome, human prostate cancer subproteome, human stomach cancer subproteome, human colon / rectal cancer subproteome, human breast cancer subproteome, human endometrial cancer subproteome, human ovarian cancer subproteome, human cervical cancer subproteome, human kidney cancer subproteome, human urinary and bladder cancer subproteome, human melanoma subproteome), (or e.g. the human type I diabetes subproteome, the human type II diabetes subproteome, Alzheimer's disease subproteome, human Parkinson's disease subproteome, human dementia subproteome, human cardiovascular disease subproteome, human down syndrome subproteome, human aging subproteome), viral proteome (e.g. human papilloma virus (HPV) proteome, human immunodeficiency virus (HIV) proteome, Orthomyxoviridae proteome, such as influenza proteome, such as Influenza A proteome, the Influenza A subtype H1N1 proteome, Influenza B proteome, Influenza C proteome or Influenza D proteome, Epstein Barr proteome, Ebolavirus proteome, Rabies lyssavirus proteome, Coronovirus proteome, such as SARS-CoV-2 (Covid-19) proteome, the SARS-CoV proteome, or the MERS-CoV, Novovirus proteome, Hepatitis A proteome, Hepatitis B proteome, Hepatitis C proteome, Hepatitis E proteome, Hepatitis delta proteome, Herpesvirus proteome, Papillomavirus proteome, rhinovirus proteome, Measles virus proteome, Mumps virus proteome, Poliovirus proteome, rabies proteome, rotavirus proteome, west nile virus proteome, yellow fever virus proteome, Zika virus proteome, Caudovirales proteome, Nimaviridae proteome, Riboviria proteome, Inoviridae proteome, Fuselloviridae proteome, Herpesvirales proteome, Asfarviridae proteome, Bicaudaviridae proteome, tuberculosis proteome, bovine tuberculosis proteome), zoonotic virus proteome, bacterial proteome (e.g. Escherichia coli (E. coli) proteome, Pseudomonas aeruginosa (P. aeruginosa) proteome, Salmonella proteome, Staphylococcus aureus proteome, Acinetobacter baumannii proteome, Bacteroides fragilis proteome, Burkholderia cepacia proteome, Clostridium difficile proteome, Clostridium sordellii proteome, Enterobacteriaceae proteome, Enterococcus faecalis proteome, Klebsiella pneumoniae proteome, Methicillin-resistant Staphylococcus aureus proteome, Morganella morganii proteome, Mycobacterium proteome, such as the Mycobacterium tuberculosis proteome), parasitic proteome (e.g. Plasmodium proteome, Toxoplasma gondii proteome, Trichomonas vaginalis proteome, Giardia duodenalis proteome, Cryptosporidiu proteome or any combination thereof. In some embodiments, the Plasmodium proteome is the Plasmodium falciparum proteome, Plasmodium knowlesi proteome, Plasmodium malariae proteome, Plasmodium ovale proteome or Plasmodium vivax proteome) and any combination thereof.Amino acid types

[0155] In the methods described, three or more amino acid types are labelled.

[0156] All amino acids have a common structure: a carboxylic acid, an amine, and an alpha carbon which has an R-group side chain. The carboxylic acid, amine, and alpha carbon are common to all amino acid types. Within chains of amino acids (peptides, oligopeptides, polypeptides, proteins), peptide bonds, which are a type of amide bonds, link adjacent amino acids. These adjacent amino acids have undergone a condensation reaction in which the non-side chain carboxylic acid group of one amino acid reacted with the non-side chain amine group of the other. One adjacent amino acid has lost a hydrogen and oxygen from its carboxyl group (COOH) and the other has lost a hydrogen from its amine group (NH 2 ), producing a molecule of water (H 2 O) and two amino acids joined by a peptide bond (-CO-NH-). Amino acids joined in this way can also be called residues or amino acid residues. All amino acids participate in the peptide backbone, which describes the repetitive covalent linkages from one amino acid to the next which incorporates the amine nitrogen, alpha carbon, and carboxyl carbon of each amino acid linked via a peptide bond to the same atoms of the next amino acid in a repeating pattern. Every alpha carbon has a variable side chain, called an R-group, which does not participate in the peptide backbone. An amino acid type is defined by the R-group, i.e. side chain. The R-group is specific to each amino acid type. The R-group of one amino acid type is distinguishable from the R-group of every other amino acid type. For example, the R-group for lysine (K) is a ε-primary amino group. Every K amino acid has this ε-primary amino group when translated. Therefore, the K amino acid type is defined by the ε-amino R-group. In another example, the R-group for tryptophan (W) is an indole group. Every W amino acid has an indole group. Therefore, the W amino acid type is defined by the indole R-group. Hence, the amino acid type K is distinguishable to the amino acid type W because of the different R-groups between these amino acid types. If the R-group of an amino acid type is subsequently modified after translation, such as post-translationally modified, the amino acid type does not change.

[0157] Thethree or more amino acid types encompassed by the invention include modified and / or unmodified amino acids of each amino acid type. This includes modified and / or unmodified amino acids of the 22 proteinogenic amino acid types and / or non-proteinogenic or synthetic amino acids.

[0158] The three or more amino acid types encompassed by the invention include the 22 proteinogenic amino acids selected from: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), pyrrolysine (O), selenocysteine (U), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V), and any combination thereof.

[0159] In some embodiments, the three or more amino acid types are selected from: cysteine (C), tyrosine (Y), lysine (K), arginine (R), histidine (H), proline (P), aspartic acid (D), glutamic acid (E), asparagine (B), glutamine (Q), serine (S) and / or threonine (T) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: tryptophan (W), cysteine (C), tyrosine (Y), lysine (K), arginine (R), histidine (H), proline (P), aspartic acid (D), glutamic acid (E), asparagine (B) and / or glutamine (Q) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: tryptophan (W), cysteine (C), tyrosine (Y) and / or lysine (K) and any combination thereof. In some embodiments, the three or more amino acids are selected from: cysteine (C), arginine (R), histidine (H) and / or aspartic acid (D) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: cysteine (C), arginine (R), histidine (H) and / or glutamic acid (E) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: cysteine (C), arginine (R), histidine (H) and / or glutamine (Q) or the modified types thereof and any combination thereof. In some embodiments, the three or more amino acid types are selected from: cysteine (C), arginine (R), tryptophan (W) and / or aspartic acid (D) or the modified version thereof and any combination thereof. In some embodiments, the three or more amino acid types are selected from: lysine (K), Arginine (R), histidine (H) and / or aspartic acid (D) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: lysine (K), tryptophan (W), arginine (R) and / or glutamic acid (E) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: tyrosine (Y), lysine (K), cysteine (C) and / or aspartic acid (D) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: tyrosine (Y), lysine (K), cysteine (C) and / or glutamic acid (E) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: proline (P), cysteine (C), arginine (R), and / or glutamic acid (E) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: proline (P), cysteine (C), arginine (R) and / or aspartic acid (D) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: cysteine (C), asparagine (B), arginine (R) and / or aspartic acid (D) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: cysteine (C), asparagine (B), arginine (R) and / or glutamic acid (E) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: lysine (K), asparagine (B), tryptophan (W) and / or cysteine (C) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), histidine (H), proline (P) and / or aspartic acid (D) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), lysine (K), cysteine (C) and / or aspartic acid (D) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), lysine (K), cysteine (C) and / or glutamic acid (E) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), lysine (K), cysteine (C) and / or tryptophan (W) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), lysine (K), cysteine (C) and / or tyrosine (Y) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), lysine (K), histidine (H) and / or tryptophan (W) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), lysine (K), histidine (H) and / or cysteine (C) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), lysine (K), histidine (H) and / or tyrosine (Y) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), cysteine (C), tryptophan (W) and / or tyrosine (Y) and any combination thereof. In some embodiments, the three or more amino acid types are selected from: arginine (R), cysteine (C), tryptophan (W) and / or proline (P) and any combination thereof.. In some embodiments, 3 amino acid types are labelled and the 3 amino acid types labelled are tryptophan (W), cysteine (C), and tyrosine (Y). In some embodiments, 3 amino acid types are labelled and the 3 amino acid types labelled are cysteine (C), tyrosine (Y) and lysine (K). In some embodiments, 3 amino acid types are labelled and the 3 amino acid types are tryptophan (W), cysteine (C) and lysine (K). In some embodiments, 3 amino acid types are labelled and the 3 amino acid types are lysine (K), tryptophan (W) and tyrosine (Y). In some embodiments, 3 amino acid types are labelled and the 3 amino acid types are tryptophan (W), tyrosine (Y) and cysteine (C). In some embodiments, 3 amino acid types are labelled and the 3 amino acid types are tryptophan (W), tyrosine (Y) and lysine (K). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: cysteine (C), tryptophan (W) and tyrosine (Y). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: asparagine (R), glutamic acid (E) and Glycine (G). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: alanine (A), leucine (L) and serine (S). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: asparagine (A), glutamic acid (E) and leucine (L). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: alanine (A), aspartic acid (D) and leucine (L). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: alanine (A), leucine (L) and proline (P). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: alanine (A), glutamic acid (E) and leucine (L). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: leucine (L), serine (S) and valine (S). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: glutamic acid (E), Isoleucine (I) and proline (P). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: glutamic acid (E), Glycine (G) and valine (V). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: Arginine (R), serine (S) and valine (V). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: alanine (A), leucine (L) and lysine (K). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: alanine (A), Arginine (R) and leucine (L). In some embodiments, 3 amino acid types are labelled, and the 3 amino acid types labelled are: alanine (A), leucine (L) and valine (V). In some embodiments, 4 amino acid types are labelled and the 4 amino acid types labelled are selected from the group consisting of: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic Acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), pyrrolysine (O), selenocysteine (U), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V), and any combination thereof. In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are tryptophan (W), tyrosine (Y), lysine (K) and cysteine (C). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are cysteine (C), arginine (R), Histidine (H) and aspartic acid (D). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are cysteine (C), arginine (R), histidine (H) and glutamic acid (E). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are cysteine (C), arginine (R), histidine (H) and Glutamine (Q). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are cysteine (C), arginine (R), tryptophan (W) and aspartic acid (D). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are lysine (K), arginine (R), histidine (H) and aspartic acid (D). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are lysine (K), tryptophan (W), arginine (R) and glutamic acid (E). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are tyrosine (Y), lysine (K), cysteine (C) and aspartic acid (D). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are tyrosine (Y), lysine (K), cysteine (C) and glutamic acid (E). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are proline (P), cysteine (C), arginine (R), and glutamic acid (E). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are proline (P), cysteine (C), arginine (R) and aspartic acid (D). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are cysteine (C), asparagine (B), arginine (R) and aspartic acid (D). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are cysteine (C), asparagine (B), arginine (R) and glutamic acid (E). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are lysine (K), asparagine (B), tryptophan (W) and cysteine (C). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), histidine (H), proline (P) and aspartic acid (D). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), lysine (K), cysteine (C) and aspartic acid (D). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), lysine (K), cysteine (C) and glutamic acid (E). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), lysine (K), cysteine (C) and tryptophan (W). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), lysine (K), cysteine (C) and tyrosine (Y). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), lysine (K), histidine (H) and tryptophan (W). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), lysine (K), histidine (H) and cysteine (C). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), lysine (K), histidine (H) and tyrosine (Y). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), cysteine (C), tryptophan (W) and tyrosine (Y). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), cysteine (C), tryptophan (W) and proline (P). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are Glutamine (Q), leucine (L), lysine (K) and valine (V). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are arginine (R), Isoleucine (I), leucine (L) and serine (S). In some embodiments, 4 amino acid types are labelled, and the 4 amino acid types labelled are alanine (A), asparagine (N), glutamic acid (E), and serine (S). In some embodiments, 5 amino acid types are labelled and the 5 amino acid types labelled are arginine (R), glutamic acid (E), lysine (K), serine, and Glutamine (Q) In some embodiments, 5 amino acid types are labelled and the 5 amino acid types labelled are arginine (R), aspartic acid (D), lysine (K), serine, and Glutamine (Q) In some embodiments, 5 amino acid types are labelled and the 5 amino acid types labelled are arginine (R), glycine (G), lysine (K), serine, and Glutamine (Q) In some embodiments, 5 amino acid types are labelled and the 5 amino acid types labelled are alanine (A), aspartic acid (D), glycine (G), serine, and arginine (R) In some embodiments, 5 amino acid types are labelled and the 5 amino acid types labelled are pyrrolysine (O), aspartic acid (D), glycine (G), serine, and arginine (R) In some embodiments, 5 amino acid types are labelled and the 5 amino acid types labelled are pyrrolysine (O), aspartic acid (D), Selenocysteine (U), serine, and arginine (R). In some embodiments, 5 amino acid types are labelled and the 5 amino acid types labelled are pyrrolysine (O), aspartic acid (D), selenocysteine (U), lysine, and arginine (R).

[0160] The amino acid types encompass L (levo) isomers and / or D (dextro) isomers of each amino acid type.

[0161] In some embodiments, the three or more labelled amino acid types comprise modified amino acids and / or unmodified amino acids of an amino acid type. In some embodiments, an amino acid type comprises the unmodified amino acids of an amino acid type. In some embodiments, the unmodified amino acids of an amino acid type have not undergone post-translational modification. In some embodiments, an amino acid type comprises the modified amino acids of an amino acid type. In some embodiments, the modified amino acids of an amino acid type have undergone post-translational modification. In some embodiments, an amino acid type comprises the modified and unmodified amino acids of an amino acid type. For example, the amino acid type cysteine (C) can comprise unmodified cysteine amino acids (C R ), modified cysteine amino acids such as cysteine disulfide (C D ) and / or a combination of both the unmodified and cysteine disulphide amino acids of cysteine. In some embodiments, a modification of amino acids, such as a post-translational modification, occurs on or including an amino acid R-group. In some embodiments, after a modification, the modified R-groups are not available for a labelling reaction. In some embodiments, the unmodified amino acid type are the amino acids within the amino acid type whose R-groups have not been modified and are therefore available for labelling without any prior chemical modifications. In some embodiments, the modified amino acids are the amino acids within the amino acid type whose R-groups have been modified and are not available for labelling without any prior chemical modifications.

[0162] In some embodiments, the amino acid type alanine (A) refers to unmodified alanine amino acids, modified alanine amino acids and / or a combination of modified and unmodified alanine amino acids. In some embodiments, the amino acid type arginine (R) refers to unmodified arginine amino acids, modified arginine amino acids and / or a combination of modified and unmodified arginine amino acids. In some embodiments, the amino acid type, asparagine (N) refers to unmodified asparagine amino acids, modified asparagine amino acids and / or a combination of modified and unmodified asparagine amino acids. In some embodiments, the amino acid type aspartic acid (D) refers to unmodified aspartic acid amino acids, modified aspartic acid amino acids and / or a combination of modified and unmodified aspartic acid amino acids. In some embodiments, the amino acid type cysteine (C) refers to unmodified cysteine amino acids, modified cysteine amino acids and / or a combination of modified and unmodified cysteine amino acids. In some embodiments, the amino acid type glutamic acid (E) refers to unmodified glutamic acid amino acids, modified glutamic acid amino acids and / or a combination of modified and unmodified glutamic acid amino acids. In some embodiments, the amino acid type glutamine (Q) refers to unmodified glutamine amino acids, modified glutamine amino acids and / or a combination of modified and unmodified glutamine amino acids. In some embodiments, the amino acid type glycine (G) refers to unmodified glycine amino acids, modified glycine amino acids and / or a combination of modified and unmodified glycine amino acids. In some embodiments, the amino acid type histidine (H) refers to unmodified histidine amino acids, modified histidine amino acids and / or a combination of modified and unmodified histidine amino acids. In some embodiments, the amino acid type isoleucine (I) refers to unmodified isoleucine amino acids, modified isoleucine amino acids and / or a combination of modified and unmodified isoleucine amino acids. In some embodiments, the amino acid type leucine (L) refers to unmodified leucine amino acids, modified leucine amino acids and / or a combination of modified and unmodified leucine amino acids. In some embodiments, the amino acid type lysine (K) refers to unmodified lysine amino acids, modified lysine amino acids and / or a combination of modified and unmodified lysine amino acids. In some embodiments, the amino acid type methionine (M) refers to unmodified methionine amino acids, modified methionine amino acids and / or a combination of modified and unmodified methionine amino acids. In some embodiments, the amino acid type phenylalanine (F) refers to unmodified phenylalanine amino acids, modified phenylalanine amino acids and / or a combination of modified and unmodified phenylalanine amino acids. In some embodiments, the amino acid type pyrrolysine (O) refers to unmodified pyrrolysine amino acids, modified pyrrolysine amino acids and / or a combination of modified and unmodified pyrrolysine amino acids. In some embodiments, the amino acid type proline (P) refers to unmodified proline amino acids, modified proline amino acids and / or a combination of modified and unmodified proline amino acids. In some embodiments, the amino acid type selenocysteine (U) refers to unmodified selenocysteine amino acids, modified selenocysteine amino acids and / or a combination of modified and unmodified selenocysteine amino acids. In some embodiments, the amino acid type serine (S) refers to unmodified serine amino acids, modified serine amino acids and / or a combination of modified and unmodified serine amino acids. IN some embodiments, the amino acid type threonine (T) refers to unmodified threonine amino acids, modified threonine amino acids and / or a combination of modified and unmodified threonine amino acids. In some embodiments, the amino acid type tryptophan (W) refers to unmodified tryptophan amino acids, modified tryptophan amino acids and / or a combination of modified and unmodified tryptophan amino acids. In some embodiments, the amino acid type tyrosine (Y) refers to unmodified tyrosine amino acids, modified tyrosine amino acids and / or a combination of modified and unmodified tyrosine amino acids. In some embodiments, the amino acid type valine (V) refers to unmodified valine amino acids, modified valine amino acids and / or a combination of modified and unmodified valine amino acids.

[0163] In preferred embodiments, the reactivity of the R-groups with the specific dyes disclosed in Table 3 defines whether, if an amino acid within an amino acid type has undergone a post-translational modification, the labelling reaction will label amino acid within that amino acid type that have not undergone the post-translational modification (unmodified amino acids), or will also label amino acids within that amino acid type that have undergone the post-translational modification (modified amino acids). For example, the skilled person will appreciate that if the labelling reaction involves attack of a nucleophilic R-group, such as lysine primary amine, on an electrophilic dye, the labelling reaction will not proceed if lysine has been post-translationally modified such that it no longer has a nucleophilic primary amine. As another example, the skilled person will appreciate that if the labelling reaction involves radical reaction with the tryptophan indole R-group and trichloroethanol (TCE), this reaction is not inhibited if the tryptophan indole R-group has been mono-oxidized to include a hydroxyl group. Applying these principles, whether a label discussed herein will label unmodified amino acids or almost modified amino acids that have undergone a post-translational modification available for the amino acid type is provided in the following Table (Table 1). If the labelling reaction will only label amino acids within the indicated amino acid type that are unmodified with the indicated post-translational modification, "unmodified" is shown in the labelling column. If the labeling reaction will also label amino acids within the indicated amino acid type that are modified with the indicated post-translational modification, "unmodified + modified) is shown in the labeling column. Table 1: Labelling modified and / or unmodified amino acids of each amino acid type Amino Acid TypeR-groupPTMLabelingAlanine, AmethylN-acetylationunmodified + modifiedArginine, Raliphatic guanidino groupmethylationunmodifieddeimination to citrullineunmodifiedAsparagine, Nβ-carboxamidedeamidation to D or iso(D)unmodifiedN-linked glycosylationunmodifiedAsparatic acid, Dβ-carboxylic acidisomerization to isoaspartic acidunmodified + modifiedCysteine, Cthioldisulfide-bond formationunmodifiedoxidation to sulfenic, sulfinic or sulfonic acidunmodifiedpalmitoylationunmodifiedN-acetylation (N-terminus)unmodified + modifiedS-nitrosylationunmodifiedGlutamic acid, Eγ-carboxylic acidcyclization to Pyroglutamic acid (N-terminus)unmodifiedgamma-carboxylationunmodifiedGlutamine, Qγ-carboxamidecyclization to Pyroglutamic acid (N-terminus)unmodifieddeamidation to Glutamic acidunmodifiedisopeptide bond formation to a lysineunmodifiedGlycine, GhydrogenN-Myristoylation (N-terminus)unmodified + modifiedN-acetylation (N-terminus)unmodified + modifiedHistidine, HimidazolePhosphorylationunmodifiedLysine, Kε-primary amino groupacetylationunmodifiedUbiquitinationunmodifiedSUMOylationunmodifiedmethylationunmodifiedisopeptide bond formation to a glutamineunmodifiedhydroxylationunmodifiedMethionine, MS-methyl thioetherN-acetylation (N-terminus)unmodified + modifiedN-linked Ubiquitinationunmodifiedoxidation to sulfoxide or sulfoneunmodifiedProline, PpyrrolidinehydroxylationunmodifiedSerine, ShydroxymethylPhosphorylationunmodifiedO-linked glycosylationunmodifiedN-acetylation (N-terminus)unmodified + modifiedThreonine, ThydroxylPhosphorylationunmodifiedO-linked glycosylationunmodifiedN-acetylation (N-terminus)unmodified + modifiedTryptophan, Windolemono- or di-oxidationunmodified + modifiedformation of KynurenineunmodifiedTyrosine, Yphenolsulfationunmodified + modifiedphosphorylationunmodified + modifiedValine, VisopropylN-acetylation (N-terminus)unmodified + modified

[0164] In some embodiments, if the user wishes to, the user can select whether to label only unmodified, and / or unmodified + modified versions of an amino acid type by transforming the modified amino acids of an amino acid type (e.g. by a chemical modification) into the unmodified amino acids to enable detection of both the modified and unmodified amino acids of an amino acid type. For example, when a combination of both the modified and unmodified amino acids of cysteine are being labelled , the modified amino acids (C D ) are first reduced to become unmodified cysteine amino acids (C R ) and all of the unmodified amino acids (which includes the newly reduced modified amino acids) are then labelled. The amino acids of the amino acid type cysteine (C) can undergo reversible post-translational modification (PTM). Specifically, the oxidation of cysteine amino acids into a disulphide bond during PTM is reversible. As another example, glycosylated (modified) serine, threonine, or asparagine residues can be converted to unmodified serine, thereonine, or asparagine residues by raising the pH of the sample solution, for example to pH 10.5. Glycosylation of serine, threonine, and asparagine residues is also reversible.

[0165] Cysteine disulphide (C D ) are modified cysteine amino acids. Unmodified cysteine amino acids are reduced cysteine (C R ). In some embodiments, the term cysteine (C) refers to the unmodified amino acids, i.e. reduced cysteine (C R ). In some embodiments, the term cysteine (C) refers to the modified amino acids, i.e. cysteine disulphide (C D ). In some embodiments, the term cysteine refers to both the unmodified amino acids (C R ) and the modified amino acids (C D ). In some embodiments, both the unmodified amino acids (C R ) and the modified amino acids (C D ) can both be labelled separately as part of the methods of the invention. The modified amino acids can be an amino acid type and / or the unmodified amino acids can be an amino acid type. The combination of the modified amino acids and the unmodified amino acids can also be an amino acid type.

[0166] In some embodiments, the term cysteine (C) refers to the combination unmodified cysteine amino acids, i.e. reduced cysteine (C R ) and modified cysteine amino acids, i.e. cysteine disulphide (C D ), when the modified cysteine amino acids, i.e. cysteine disulphide (C D ) has been reduced, and all of the unmodified amino acids (which includes the newly reduced modified amino acids) are then labelled. In some embodiments, the term cysteine refers to both unmodified amino acids (C R ) being labelled, and the combination of modified and unmodified amino acids when the modified amino acids have been reduced.

[0167] Unmodified amino acids of cysteine, i.e. reduced cysteine (C R ) and / or modified amino acids of cysteine, i.e. cysteine disulphide (C D ) and / or the combination of modified and unmodified amino acids of cysteine are a subset of the amino acid type cysteine (C). The unmodified amino acids of cysteine, i.e. reduced cysteine (C R ) and the combination of modified and unmodified cysteine, once the modified cysteine has been reduced can both be labelled and provide different measurements of the label. For example, both the unmodified amino acids C R and the combination of C R and C D can both be labelled with a fluorogenic dye and provide a different fluorescence intensity. Therefore, the invention encompasses reduced cysteine (C R ), cysteine disulphide (C D ) and / or the combination of modified and unmodified cysteine amino acids of the amino acid type cysteine (C). Any reference to the amino acid type cysteine (C) encompasses reduced cysteine (C R ), cysteine disulphide (C D ) and / or the combination of modified and unmodified cysteine amino acids. Preferably, any reference to the amino acid type cysteine (C) encompasses reduced cysteine (C R ) and / or the combination of modified and unmodified cysteine (C T ). Preferably, reduced cysteine (C R ) and / or the combination of modified and unmodified cysteine are labelled in the sample.

[0168] Any other amino acid types with a distinct R-group which can be labelled can equally be used as part of the invention. For example, the three or more amino acid types encompassed by the invention also includes synthetic amino acid types. Synthetic amino acid types are non-proteinogenic amino acids that occur naturally, or are chemically synthesized. Synthetic amino acid types encompassed by the invention include amino acid types which contain the functional groups azide, alkyne, alkene, cyclooctyne, diene, acyl, iodo, boronic acid, diazirine, cyclooctene, epoxide, cyclopropane, biotin, dienophile, sulfonic acid, sulfinic acid, biotin, oxime, nitrone, norbornene, tetrazene, tetrazole, quadricyclane, electron poor pi systems, electron rich pi systems, halogen, NHS ester, maleimide, and / or diazo and any combination thereof. These functional groups are incorporated in place of the natural functional groups. In addition, synthetic amino acid types encompassed by the invention also include amino acid types with synthetic substituents appended or attached to the natural functional groups of an amino acid type. For example, the invention encompasses a tryptophan amino acid which has been synthetically modified to contain a norbornene on its indole ring. In some embodiments, when the synthetic substituents are appended or attached to the natural functional groups of an amino acid type, this incorporation has taken place prior to the labelling reactions disclosed herein.Labelling three or more amino acid types

[0169] The amino acids of three or more amino acid types are labelled in the sample.

[0170] In some embodiments, the labelling reactions are specific for each amino acid type. All amino acids within every amino acid type are contained within intact protein molecules. This allows reaction with exclusively the amino acid types of interest within intact protein chains, without requiring hydrolysis of the protein chain into individual amino acids or proteolytic digestion of the protein chain into fragments containing only one or a fraction of amino acid types contained within the intact protein chain. This is similar to how an antibody reacts only with a protein of interest, even though other proteins not of interest are also present within the solution. Because of the complementary chemical reactivity of the labels and the amino acid types, the labels react exclusively with the amino acid type of interest. In some embodiments, each label reacts with only one amino acid type. In some embodiments, each label reacts with one or two amino acid types. In some embodiments, each label reacts with one, two or three amino acid types. For example, the label o-maleimide-BODIPY is specific for the cysteine (C) amino acid type because only the thiol which defines the cysteine (C) R-group can react with the maleimide moiety. This is because thiols are "soft" nucleophiles and react preferentially with "soft" electrophiles such as maleimide.

[0171] In some embodiments, each amino acid type has a distinct label for identification. For example, if 5 amino acid types are labelled, then there are 5 different labels. For example, the amino acids of the amino acid type K are labelled with a first label, and the amino acids of the amino acid type W are labelled with a second label, which is distinct from the first label.

[0172] In some embodiments, 3 amino acid types are labelled. In some embodiments, 4 amino acid types are labelled. In some embodiments, 5 amino acid types are labelled. In some embodiments, 6 amino acid types are labelled. In some embodiments, 7 amino acid types are labelled. In some embodiments, 8 amino acid types are labelled. In some embodiments, 9 amino acid types are labelled. In some embodiments, 10 amino acid types are labelled. In some embodiments, 11 amino acid types are labelled. In some embodiments, 12 amino acid types are labelled. In some embodiments, 13 amino acid types are labelled. In some embodiments, 14 amino acid types are labelled. In some embodiments, 15 amino acid types are labelled. In some embodiments, 16 amino acid types are labelled. In some embodiments, 17 amino acid types are labelled. In some embodiments, 18 amino acid types are labelled. In some embodiments, 19 amino acid types are labelled. In some embodiments, 20 amino acid types are labelled. In some embodiments, 21 amino acid types are labelled. In some embodiments, 22 amino acid types are labelled. In some embodiments, 23 amino acid types are labelled. In some embodiments, 24 amino acid types are labelled. In some embodiments, 25 amino acid types are labelled. In some embodiments, 26 amino acid types are labelled. In some embodiments, 27 amino acid types are labelled. In some embodiments, 28 amino acid types are labelled. In some embodiments, 29 amino acid types are labelled. In some embodiments, 30 amino acid types are labelled. In some embodiments, 31 amino acid types are labelled. In some embodiments, 32 amino acid types are labelled. In some embodiments, 33 amino acid types are labelled. In some embodiments, 34 amino acid types are labelled. In some embodiments, 35 amino acid types are labelled. In some embodiments, 36 amino acid types are labelled. In some embodiments, 37 amino acid types are labelled. In some embodiments, 38 amino acid types are labelled. In some embodiments, 39 amino acid types are labelled. In some embodiments, 40 amino acid types are labelled. In some embodiments, 2, 3, 4 or 5 amino acid types are labelled. In some embodiments, 4 or 5 amino acid types are labelled. In some embodiments, 3 or 4 amino acid types are labelled.

[0173] In some embodiments, the 3 amino acid types labelled are selected from: tryptophan (W), cysteine (C), tyrosine (Y) or lysine (K). In some embodiments, the 3 amino acid types labelled are: tryptophan (W), cysteine (C) and lysine (K). In some embodiments, the 3 amino acid types labelled are: lysine (K), tryptophan (W) and tyrosine (Y). In some embodiments, the 3 amino acid types labelled are: tryptophan (W), tyrosine (Y) and cysteine (C). In some embodiments, the 3 amino acid types labelled are: tryptophan (W), tyrosine (Y) and lysine (K). In some embodiments, the 3 amino acid types labelled are: cysteine (C), tryptophan (W) and tyrosine (Y). In some embodiments, the 3 amino acid types labelled are: asparagine (R), glutamic acid (E) and glycine (G). In some embodiments, the 3 amino acid types labelled are: alanine (A), leucine (L) and serine (S). In some embodiments, the 3 amino acid types labelled are: asparagine (A), glutamic acid (E) and leucine (L). In some embodiments, the 3 amino acid types labelled are: 3 amino acid types labelled are: alanine (A), aspartic acid (D) and leucine (L). In some embodiments, the 3 amino acid types labelled are: the 3 amino acid types labelled are: alanine (A), leucine (L) and proline (P). In some embodiments, the 3 amino acid types labelled are: alanine (A), glutamic acid (E) and leucine (L). In some embodiments, the 3 amino acid types labelled are: leucine (L), serine (S) and valine (S). In some embodiments, the 3 amino acid types labelled are: glutamic acid (E), isoleucine (I) and proline (P). In some embodiments, the 3 amino acid types labelled are: glutamic acid (E), glycine (G) and valine (V). In some embodiments, the 3 amino acid types labelled are: arginine (R), serine (S) and valine (V). In some embodiments, the 3 amino acid types labelled are: alanine (A), leucine (L) and lysine (K). In some embodiments, the 3 amino acid types labelled are: alanine (A), arginine (R) and leucine (L). In some embodiments, the 3 amino acid types labelled are: alanine (A), leucine (L) and valine (V).

[0174] In some embodiments, the 4 amino acid types labelled are: tryptophan (W), tyrosine (Y) and lysine (K) and cysteine (C), wherein the combination of modified and unmodified amino acids of cysteine are labelled. In some embodiments, the 4 amino acid types labelled are: tryptophan (W), cysteine (C), tyrosine (Y) and lysine (K), wherein reduced cysteine (C R ) is labelled. In some embodiments, the 4 amino acid types labelled are: tryptophan (W), cysteine (C), tyrosine (Y) and lysine (K). In some embodiments, the 4 amino acid types labelled are: cysteine (C), arginine (R), histidine (H) and aspartic acid (D). In some embodiments, the 4 amino acid types labelled are: cysteine (C), arginine (R), histidine (H) and glutamic acid (E). In some embodiments, the 4 amino acid types labelled are: cysteine (C), arginine (R), histidine (H) and Glutamine (Q). In some embodiments, the 4 amino acid types labelled are: cysteine (C), arginine (R), tryptophan (W) and aspartic acid (D). In some embodiments, the 4 amino acid types labelled are: lysine (K), arginine (R), histidine (H) and aspartic acid (D). In some embodiments, the 4 amino acid types labelled are: lysine (K), tryptophan (W), arginine (R) and glutamic acid (E). In some embodiments, the 4 amino acid types labelled are: tyrosine (Y), lysine (K), cysteine (C) and aspartic acid (D). In some embodiments, the 4 amino acid types labelled are: tyrosine (Y), lysine (K), cysteine (C) and glutamic acid (E). In some embodiments, the 4 amino acid types labelled are: proline (P), cysteine (C), arginine (R), and glutamic acid (E). In some embodiments, the 4 amino acid types labelled are: proline (P), cysteine (C), arginine (R) and aspartic acid (D). In some embodiments, the 4 amino acid types labelled are: cysteine (C), asparagine (B), arginine (R) and aspartic acid (D). In some embodiments, the 4 amino acid types labelled are: cysteine (C), asparagine (B), arginine (R) and glutamic acid (E). In some embodiments, the 4 amino acid types labelled are: lysine (K), asparagine (B), tryptophan (W) and cysteine (C). In some embodiments, the 4 amino acid types labelled are: arginine (R), histidine (H), proline (P) and aspartic acid (D). In some embodiments, the 4 amino acid types labelled are: arginine (R), lysine (K), cysteine (C) and aspartic acid (D). In some embodiments, the 4 amino acid types labelled are: arginine (R), lysine (K), cysteine (C) and glutamic acid (E). In some embodiments, the 4 amino acid types labelled are: arginine (R), lysine (K), cysteine (C) and tryptophan (W). In some embodiments, the 4 amino acid types labelled are: arginine (R), lysine (K), cysteine (C) and tyrosine (Y). In some embodiments, the 4 amino acid types labelled are: arginine (R), lysine (K), histidine (H) and tryptophan (W). In some embodiments, the 4 amino acid types labelled are: arginine (R), lysine (K), histidine (H) and cysteine (C). In some embodiments, the 4 amino acid types labelled are: arginine (R), lysine (K), histidine (H) and tyrosine (Y). In some embodiments, the 4 amino acid types labelled are: arginine (R), cysteine (C), tryptophan (W) and tyrosine (Y). In some embodiments, the 4 amino acid types labelled are: arginine (R), cysteine (C), tryptophan (W) and proline (P). In some embodiments, the 4 amino acid types labelled are: Glutamine (Q), leucine (L), lysine (K) and valine (V). In some embodiments, the 4 amino acid types labelled are: arginine (R), isoleucine (I), leucine (L) and serine (S). In some embodiments, the 4 amino acid types labelled are: alanine (A), asparagine (N), glutamic acid (E), and serine (S).

[0175] Each amino acid type refers to the modified and / or unmodified amino acids of that amino acid type. Preferably, the amino acid cysteine (C) refers to the unmodified amino acids (C R ) and / or the combination of the unmodified and the modified (cysteine disulphide) amino acids, once the modified amino acids have been reduced.

[0176] In some embodiments, the 5 amino acids types labelled are: tryptophan (W), cysteine (C), tyrosine (Y) and lysine (K), wherein both reduced cysteine (C R ), and the combination of modified (C D ) and unmodified (C R ) amino acids of cysteine are labelled. In some embodiments, the 5 amino acids types labelled are: arginine (R), glutamic acid (E), lysine (K), serine, and glutamine (Q). In some embodiments, the 5 amino acids types labelled are: arginine (R), aspartic acid (D), lysine (K), serine, and glutamine (Q). In some embodiments, the 5 amino acids types labelled are: arginine (R), glycine (G), lysine (K), serine, and glutamine (Q). In some embodiments, the 5 amino acids types labelled are: alanine (A), aspartic acid (D), glycine (G), serine, and arginine (R). In some embodiments, the 5 amino acids types labelled are: pyrrolysine (O), aspartic acid (D), glycine (G), serine, and arginine (R). In some embodiments, the 5 amino acids types labelled are: pyrrolysine (O), aspartic acid (D), selenocysteine (U), serine, and arginine (R). In some embodiments, the 5 amino acids types labelled are: pyrrolysine (O), aspartic acid (D), selenocysteine (U), lysine, and arginine (R).

[0177] In some embodiments, the three or more amino or more acid types can be labelled with the same label and the label is independently identified for each amino acid type. For example, the amino acids of the amino acid type W are labelled with the same label as the amino acids of the amino acid type Y and the label of the amino acid type W is independently identified to the label of the amino acid type Y. In some embodiments, when three amino acid types are labelled with the same label, the parameters for detecting the label are distinct. For example, the label for one amino acid type is deconvoluted from the label for a second amino acid type. For example, the amino acid types of tryptophan (W) and tyrosine (Y) can both be labelled with the same fluorescent label, but the fluorescence intensity of the tryptophan (W) label is deconvoluted from the fluorescence intensity of the tyrosine (Y) label. In some embodiments, the amino acid types of tryptophan (W) and tyrosine (Y) are both labelled with the same fluorogenic dye, but the excitation and emission wavelengths for measuring the signal from the fluorogenic dye for tryptophan (W) are different than the excitation and emission wavelength parameters for measuring the signal from the fluorogenic dye for tyrosine (Y). In some embodiments, the amino acid types of tryptophan (W) and tyrosine (Y) are both labelled with the same fluorogenic dye, but the excitation and emission wavelengths for measuring the signal from the fluorogenic dye for tryptophan (W) are different from the excitation and emission wavelengths for measuring the signal from the fluorogenic dye for tyrosine (Y) and tryptophan (W). In some embodiments, the tyrosine (Y) signal is measured from the total tryptophan (W) and tyrosine (Y) signal minus the tryptophan signal (W).

[0178] In some embodiments, the three or more amino acid types can be labelled (e.g. reacted) with the same label but the labelling (e.g. reactions) are performed under different conditions. In some embodiments, a multi-step labelling process allows the same label to react specifically with only one amino acid type. For example, methionine (M) and phenylalanine (F) amino acid types can be reacted with the same label, a dye bearing an azide reactive group. The labelling reaction involves Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), also known as "click chemistry". The first step of the labelling reaction for the methionine (M) or phenylalanine (F) amino acid types is installation of an alkyne group onto the methionine (M) or phenylalanine (F) R-group that subsequently reacts with the azide on the dye during the second step of the labelling reaction. This first step is performed under conditions specific for reaction with only the methionine (M) amino acid type or only the phenylalanine (F) amino acid type. In this way, the same label (e.g. dye) can react specifically with more than one amino acid type, such that only the desired amino acid type is labelled under the conditions of the reaction.

[0179] In some embodiments, all of the three or more amino acid types which are labelled are labelled within the whole sample. In some embodiments, the sample is not separated into multiple individual fractions prior to the labelling reaction. For example, a urine sample is provided and the amino acid types W, Y and K are all labelled in the urine sample, without separating the sample into multiple individual fractions, and labelling W, Y and K separately in separate fractions. For example, a single protein molecule will have all of the three or more amino acid types labelled within the molecule. In some embodiments, all of the amino acid types being labelled are labelled in one fraction. In this embodiment, the label of each amino acid type is selected to be specific to one amino acid type so that it does not cross react with the other amino acid type. In some embodiments, the selection of the label is governed by the chemistry of the amino acid type to be modified. For example, when lysine and tryptophan are labelled in the same fraction, the labelling chemistries do not interfere with one another, and the signal of the dye linked to tryptophan is separable from the signal of the dye linked to lysine, i.e. different excitation and emission wavelengths in the case of fluorescence intensity.

[0180] In some embodiments, the sample is separated into multiple fractions prior to the labelling reaction. Because the amino acids of each amino acid type are contained within intact protein molecules which are not hydrolysed or digested, one protein molecule contains many amino acid types, and therefore one fraction contains many amino acid types. When the sample is separated into multiple fractions, different labelling reactions are performed in each fraction which label specifically the amino acid type of interest. In some embodiments, each fraction contains an equal volume. In this embodiment, each fraction is labelled. For example, the sample is separated into two fractions before labelling and 4 amino acid types are being labelled; wherein two amino acid types are labelled in one fraction and two alternative amino acid types are labelled in the second fraction. For example, the 4 amino acid types in the sample being labelled are W, K, Y and C, wherein C is the combination of C D and C R . The sample is separated into two fractions before labelling; in the first fraction, the amino acid types (W) and lysine (K) are labelled with using labels specific for the (W) and (K) amino acid types, and in the second fraction the amino acid types cysteine (C) and tyrosine (Y) are labelled using labels specific for the (C) and (Y) amino acid types. In another example, the sample is separated into four fractions before labelling. 4 amino acid types are being labelled; with one amino acid type being labelled in each fraction. For example, the 4 amino acid types in the sample being labelled are W, K, Y and C. The sample is separated into four fractions before labelling; the amino acid type tryptophan (W) is labelled in the first fraction, the amino acid type lysine (K) is labelled in the second fraction, the amino acid type cysteine (C) is labelled in the third fraction, and the amino acid type tyrosine (Y) is labelled in the fourth fraction. In some embodiments, the number of fractions is equal to the number of amino acid types being labelled, and one amino acid type is labelled per fraction. In some embodiments, the number of fractions is less than the number of amino acid types being labelled, and more than one amino acid type is labelled per fraction. The presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest, or, a mixture of proteins, peptides, polypeptides, oligopeptides, subproteomes or proteomes of interest within a sample is determined, for each fraction, based on the measured label of each fraction.

[0181] In some embodiments, if two amino acid types have the same label, they are labelled and measured in different fractions. For example, in some embodiments, the amino acid type W and Y are labelled and measured in different fractions. In some embodiments, if the label of a first amino acid type is predicted to cross react with the label of a second amino acid type, then the first and second amino acid types are separated into separate fractions. The first fraction is reacted with a label that is specific for the first amino acid type within the sample, and the second fraction is reacted with a label that is specific for the second amino acid type within the sample. This avoids cross-reaction of the label.

[0182] In some embodiments, the three or more amino acid types to be labelled are separated into a fraction with a fluorogenic dye which does not cross-react with another fluorogenic dye or amino acid type in the sample. In some embodiments, all of the amino acids, i.e. every amino acid, of three or more amino acid types in the sample are labelled. For example, if the amino acid type tryptophan was being labelled, then every tryptophan amino acid present in the sample is labelled. In some embodiments, every amino acid (i.e. all amino acids) of each of three or more amino acid types in the sample is labelled. For example, if the three or more amino acid types to be labelled are tryptophan (W), lysine (K) and tyrosine (Y), then every, i.e. all, tryptophan (W) amino acids in the sample are labelled, every, i.e. all, lysine (K) amino acids in the sample are labelled and every, i.e. all, tyrosine (Y) amino acids in the sample are labelled. In some embodiments, a proportion of the amino acids (i.e. not all amino acids) of three or more amino acid types in the sample are labelled. In some embodiments, a proportion of amino acids (i.e. not all amino acids) of each of three or more amino acid types in the sample are labelled. For example, if the amino acid type tryptophan was being labelled, then a proportion of the tryptophan amino acids present in the sample are labelled. For example, if the three or more amino acid types to be labelled are tryptophan (W), lysine (K) and tyrosine (Y), then a proportion of tryptophan (W) amino acids in the sample is labelled, a proportion of lysine (K) amino acids in the sample is labelled and a proportion of tyrosine (Y) amino acids in the sample is labelled. Preferably, about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of amino acids of each of three or more amino acid types are labelled within the sample. In some embodiments, the proportion of the amino acid of an amino acid type labelled within the sample is determined using mass spectrometry. In some embodiments, a proportion of the amino acids (i.e. not all amino acids) of three or more amino acid types in the proteome or subproteome contained within the sample are labelled.

[0183] In some embodiments, every (i.e. all) of the amino acids of one amino acid type are labelled and a proportion of the amino acids of another amino acid type are labelled. For example, if the three or more amino acid types to be labelled include tryptophan (W) and lysine (K), then all of the tryptophan (W) amino acids in the sample and 90% of the lysine (K) amino acids in the sample are labelled. Alternatively, 90% of the tryptophan (W) amino acids in the sample and all of the lysine (K) amino acids in are labelled.

[0184] In some embodiments, the R-group of amino acids within three or more amino acid types is labelled within the sample. The R-group of each amino acid type is unique for each amino acid type. For example, the R-group of tryptophan (W) is distinct to the R-group of lysine (K). The R-group specific to each amino acid type is provided in Table 2. Three or more amino acid types in the sample are labelled. In some embodiments, every amino acid (i.e. all the amino acids) of an amino acid type selected to be labelled is labelled. In some embodiments, the R-group of every amino acid (i.e. all the amino acids) of an amino acid type are labelled. In some embodiments, a proportion (i.e. not every amino acid) of an amino acid type is labelled. In some embodiments, the R-group of a proportion of the amino acids (i.e. not all of the amino acids) of an amino acid type is labelled. In some embodiments, every amino acid (i.e. all the amino acids) of an amino acid type are labelled, and a proportion (i.e. not all of the amino acids) of a second amino acid type are labelled. In some embodiments, the R-group of every amino acid (i.e. all the amino acids) of a first amino acid type are labelled and the R-group of a proportion of the amino acids (i.e. not all of the amino acids) of a second amino acid type are labelled.

[0185] Preferably, the R-groups for each of three or more of the amino acid types selected from: W, C, Y or K are labelled. Preferably, the R-group labelled for C is the R-group of reduced cysteine (C R ). Preferably, the R group being labelled for C is the R-group of the combination of C D and C R , after C D has been reduced. Preferably, both the R-groups for (C R ) and the combination of C D and C R , after C D has been reduced, within the amino acid type cysteine (C) are labelled within the sample.

[0186] In a preferred embodiment, three or more amino acid types are labelled, and the R-groups of each of the amino acid types are labelled in the sample (i.e. three or more types of R-groups are labelled). The three or more amino acid types of R-groups corresponds to the three or more amino acid types. For example, when tryptophan and lysine are amino acid types being labelled, the R-group for tryptophan and the R-group for lysine are labelled in the sample. In some embodiments, the R-groups of each of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid types are labelled. In some embodiments, the 3 amino acid R-groups being labelled are the R-groups for each of the 3 amino acid types selected from: C, W and Y, wherein C is the unmodified C amino acids (C R ) and the combination of C D and C R , after C D has been reduced. In some embodiments, the 3 amino acid R-groups being labelled are the R-groups for each of the 3 amino acid types selected from: C W and K, wherein C is the unmodified C amino acids (C R ) and the combination of C D and C R , after C D has been reduced. In some embodiments, the 3 amino acid R-groups being labelled are the R-groups for each of the 3 amino acid types selected from: C, K and Y, wherein C is the unmodified C amino acids (C R ) and the combination of C D and C R , after C D has been reduced. In some embodiments, the 4 amino acid R-groups being labelled are the R-groups for each of the 4 or more amino acid types selected from: C, W, K and Y, wherein C is the combination of C D and C R , after C D has been reduced. In some embodiments, the 4 amino acid R-groups being labelled are the R-groups for each of the 4 amino acid types selected from: C R , K, W and Y. In some embodiments, the 4 amino acid R-groups being labelled are the R-groups for each of the 4 amino acid types selected from: C K, W and Y, , wherein C is the unmodified C amino acids (C R ) and the combination of C D and C R , after C D has been reduced.

[0187] In some embodiments, one amino acid R-group is labelled for each amino acid type. For example, the indole R-group on each tryptophan amino acid is labelled for the amino acid type tryptophan. In another example, the ε-amino R-group on each lysine amino acid is labelled for the amino acid type lysine. The R-group for each amino acid type is outlined in Table 2. Table 2: R-group for each amino acid type Amino acid type Modified or unmodified amino acids of the amino acid type R-group labelled Alanine (A)UnmodifiedMethylarginine (R)Unmodifiedaliphatic guanidino group; partial primary amine character or equal primary amine characterN-Glycosylated arginine (GR)ModifiedCarbohydrate glycoside bonded to guanidino amineasparagine (N)Unmodifiedβ-carboxamideN-Glycosylated asparagine (GN)ModifiedCarbohydrate glycoside bonded to β-carboxamide amineAspartic acid (D)Unmodifiedβ-carboxylic acidCysteine (C)Modified (C D )Oxidised (disulphide bonded) thiolCysteine (C)Unmodified (C R )Reduced thiolCysteine (C)Unmodified and modified, after modified have been reducedReduced thiolglutamic acid (E)Unmodifiedγ-carboxylic acidGlutamine (Q)Unmodifiedγ-carboxamideglycine (G)UnmodifiedHydrogenhistidine (H)UnmodifiedImidazoleIsoleucine (I)Unmodifiedsec-butylLeucine (L)UnmodifiedIsobutylLysine (K)Unmodifiedε-primary amino groupN6-(pyridoxal phosphate)lysineModifiedPyridoxyal phosphate aldimineMethionine (M)UnmodifiedS-methyl thioetherPhenylalanine (F)UnmodifiedBenzylProline (P)UnmodifiedPyrrolidine4-hydroxyproline (HP)Modified4-hydroxypyrrolidineserine (S)UnmodifiedHydroxymethylPhosphoserine (PS)ModifiedPhospho methyl esterThreonine (T)UnmodifiedHydroxylPhosphothreonine (PT)ModifiedPhosphoesterTryptophan (W)UnmodifiedIndoleTyrosine (Y)UnmodifiedPhenolPhosphothrosine (PY)ModifiedPhosphophenolvaline (V)UnmodifiedIsopropyl

[0188] In a preferred embodiment, the three or more amino acid types within the sample are labelled fluorescently, isotopically, or using mass tags. Alternatively, the three or more amino acid types within the sample are labelled with nucleotides. In some embodiments, the R-group of each amino acid type is labelled fluorescently, isotopically, or using mass tags. In some embodiments, the R-group of each amino acid is labelled with nucleotides.

[0189] In some embodiments, one amino acid is labelled with one type of label and another amino acid type is labelled with another type of label. For example, one amino acid type is labelled with a fluorescent label and a second amino acid type is labelled with a tandem mass tag.

[0190] In some embodiments, the label is a fluorescent label. In some embodiments, the fluorescent label is a fluorescent dye, fluorescent tag, fluorescent probe, or fluorescent protein. In some embodiments, the fluorescent label includes a fluorophore. In some embodiments, the fluorophore is selected from the group consisting of: Hydroxycoumarin, Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, BODIPY-FL, G-Dye100, G-Dye200, G-Dye300, G-Dye400, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), APC-Cy7 conjugates, DAPI, Hoechst 33258, SYTOX Blue, Chromomycin A3, Mithramycin, YOYO-1, ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO Rho13, ATTO 594, ATTO 610, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, Brilliant Violet 421, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 750, Brilliant Violet 785, TM-BDP, KFL-1, KFL-2, KFL-3, KFL-4, Super Bright 436, Super Bright 600, Super Bright 645, Super Bright 702, Super Bright 780, Alexa Flour 350, Alexa Flour 405, Alexa Flour 488, Alexa Flour 532, Alexa Flour 546, Alexa Flour 555, Alexa Flour 568, Alexa Flour 594, Alexa Flour 647, Alexa Flour 680, Alexa Flour 850, Coumarin, Pacific Green, Oregon Green, Flourescein (FITC), PE-Cyanine7, PerCP-Cyanine5.5, Tetramethylrhodamine (TRITC), eFlour 450, eFlour506, eFlour660, PE-eFlour 610, PerCP-eFlour 710, APC-eFlour 780, Super Bright 436, Super Bright 600, Super Bright 645, Super Bright 702, Super Bright 780, DAPI, SYTOX Green, SYTO 9, TO-PRO-3, Qdot 525, Qdot 565, Qdot 605, Qdot 655, Qdot 705, Qdot 800, R-Phycoerythrin (R-PE), , VioBlue, VioGreen, VioBright 515, Vio 515, VioBright FITC, PE, PE-Vio 615, PerCP, PerCP-Vio 700, PE-Vio 770, APC, APC-Vio 770, 1,8-Naphthalimides, Thiazole Orange, CyTRAK Orange, LDS 751, 7-AAD, SYTOX Orange, TOTO-3, TO-PRO-3, DRAQ5, DRAQ7, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, CFP, GFP (emGFP), RFP (tagRFP), GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, mCerulean, mCerulean3 mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midoriishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 ("RFP"), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum or mRaspberry.

[0191] In some embodiments, the fluorescent tag or fluorescent label is not a fluorogenic dye. In some embodiments, the fluorescent tag or fluorescent label also includes a reactive group that is specific for the R-group which defines an amino acid type. In this way, the fluorescent label targets a particular amino acid type. In some embodiments, labelling an amino acid type of interest is covalently labelling an amino acid type of interest. In some embodiments, the reactive group permits selective covalent labelling of the R-group of the amino acid type of interest. In some embodiments, the reactive group is selected from the group consisting of: NHS-ester, maleimide, alkyne, azide, bromide, chloride, fluoride, iodide, aryl bromide, aryl chloride, aryl fluoride, aryl iodide, diene, dienophile, olefin, tetrazine, cyclooctyne, biotin, streptavidin, isothiocyanate, active ester, sulfonyl chloride, dialdehyde, iodoacetamide, ethylenediamine, aminoacridone, hydrazide, carboxyl, or alkoxyamine. For example, it is appreciated by those skilled in the art that the electrophilic maleimide group selectively targets nucleophilic cysteine thiol residues. Therefore, any of the fluorophores listed above can be selected and coupled with a maleimide reactive group, to selectively label cysteine thiol resides. For example, cysteine thiol residues can be labelled with a fluorescent label comprising Super Bright 436 and a maleimide reactive group. As another example, it is appreciated by those skilled in the art that the labile NHS ester group selective targets the lysine primary amine R-group, and can undergo a covalent SN2 reaction with the lysine primary amine R-group. Therefore, the lysine residues can be labelled with the NHS-ester form of Cy5. These methods of labeling are appreciated by the skilled person and the indicated reactive forms of the fluorophores disclosed are commercially available.

[0192] In some embodiments, the fluorescent label is a fluorogenic dye which targets an amino acid type or a molecule which becomes fluorescent exclusively upon reaction with an amino acid type. Preferably, the fluorogenic dye becomes fluorescent exclusively after covalently reacting with specific amino acid types within the protein. In this case, there is no need to couple a fluorophore with a reactive group, because in the case of a fluorogenic dye or molecule which becomes fluorescent exclusively on reaction with an amino acid type, the selectivity for an amino acid type is already built into the chemical structure of the fluorogenic dye or molecule which becomes fluorescent exclusively upon reaction with an amino acid type. In some embodiments, the fluorogenic dye which targets an amino acid type or a molecule which becomes fluorescent exclusively upon reaction with an amino acid type is selected from the group consisting of: 4-Fluoro-7-sulfamoylbenzofurazan (ABD-F), 2,2,2-Trichloroethanol (TCE) and / or ortho-phthalaldehyde (OPA), or a mixture thereof. Preferably, the fluorogenic dye is selected for each amino acid type, or R-group in Table 2 and Table 3. However, this list is non-exhaustive and any other fluorogenic dye or molecule which becomes fluorescent upon reaction with an amino acid type known within the art can also be used. Those skilled in the art will appreciate that labelling with high quantum yield fluorogenic or non-fluorogenic labels can permit identification of very low concentrations of protein within the sample, such as at the single molecule level. This corresponds to protein concentrations between 1 pM and 1 nM.

[0193] In some embodiments, amino acid type is reacted with a molecule which becomes fluorescent after reaction with the amino acid type, or which shifts the fluorescence of an already fluorescent amino acid type into the visible spectrum. For example, in some embodiments, the molecule which becomes fluroescent after reaction with an amino acid type is a halo compound. In some embodiments, the halo compounds are trichloroacetic acid, chloroform, triflouroethanol, triflouroacetic acid, flouroform, tribromoethanol, tribromoacetic acid, bromoform, triiodoethanol, triiodoacetic acid or iodoform. In some embodiments, the amino acid types tryptophan (W) and / or tyrosine (Y) are labelled with Trichloroethanol trichloroethanol (TCE ), trichloroacetic acid (TCA), chloroform, trifluoroethanol (TFE), triflouroacetic acid (TFA), flouroform, tribromoethanol, tribromoacetic acid (TBA), bromoform, triiodoethanol (TIE), or triiodoacetic acid (TIA), iodoform, or, with 2-(2-(2-methoxyethoxy)ethoxy)ethyl (E)-2-diazo-4-phenylbut-3-enoate in the presence of Rh2(OAc)4, tBuHNOH. In some embodiments, the amino acid type Y is labelled with trichloroethanol (TCE), or, installation of an aryl group ortho to the tyrosine hydroxyl groups using [RhCl(PPh3)3], R2P(OAr),Ar-Br,CsCO3.

[0194] The skilled person would readily understand how amino acids can be labelled.

[0195] In some embodiments, the label is selected based on a specific interaction with an amino acid type. For example, the label is a fluorogenic dye and is selected based on a specific interaction with an amino acid type where the dye only becomes fluorescent (i.e. its signal only becomes detectable) after it has reacted with the specific amino acid type. In some embodiments, the selection of the label is governed by the chemistry of the amino acid type to be modified. In some embodiments, for specific reaction with an amino acid type, there is a reactive group on an amino acid type and a reactive group on a label that react exclusively with one another. This is determined by the specific chemical reactivity of the R-group on an amino acid type and the reactive group on a label. For example, ABD-F contains a halogen at a labile position on an aromatic system and is susceptible to electrophilic aromatic substitution. There are several nucleophilic amino acid types (e.g. cysteine, lysine, histidine), but the cysteine amino acid type (C) is the strongest nucleophile because it is the most polarizable. Because the electron cloud is more polarizable, the activation energy for nucleophilic attack is reduced. Therefore, ABD-F reacts preferentially with cysteine (C) residues and does not react with other amino acid types, such as lysine, or histidine amino acid types, which would require a higher activation energy.

[0196] In some embodiments, the labelling reaction is a fluorogenic reaction. This means that fluorescence is generated exclusively after reaction with the amino acid type, such that there is not a need to purify the unreacted label from the sample.

[0197] In some embodiments, a fluorogenic reaction involves removing a group from a fluorophore that quenches reaction. For example, it is known that maleimide quenches flourophores when it is directly conjugated to fluorophores due to maleimide's low energy nπ* state provides a non-radiative pathway for decay of the flourophore's excited state, and can also quench flourophores when it is joined to the fluorophore by a spacer group because photoinduced electron transfer (PET) to the C=C double bond can occur. For example, when maleimide is attached in the ortho position to the fluorescent dye BODIPY, maleimide quenches the fluorescence of the dye BODIPY. However, when o-maleimide BODIPY reacts with the thiol R-group of the cysteine (C) amino acid type, the C=C double bond becomes saturated and no longer quenches fluorescence, so the BODIPY label becomes emissive. Other quenching groups known in the art include azido, alkyne, phosphine, sydnone, tetrazine, or oxime and these can become unquenched after a fluorogenic click reaction, including copper-catalyzed / strain-promoted alkyne-azide cycloaddition (CuAAC / SPAAC), Staudinger ligation, copper-catalyzed / strain-promoted sydnone-alkyne cycloaddition (CuSAC / SPSAC), inverse electron demand Diels-Alder reaction (iEDDA), or 1,3- dipolar cycloaddition.

[0198] In some embodiments, a fluorogenic reaction involves generating a fluorophore. An example of this type of fluorogenic reaction is the reaction of the lysine (K) amino acid type with ortho-pthalaldehyde. A second ring is formed, extending the electronic conjugation, and this larger delocalized pi system becomes fluorescent in the visible region of the spectrum. In some embodiments, a fluorogenic reaction involves changing the fluorescence properties of an existing fluorescent substrate. For example, the amino acid tryptophan which is intrinsically fluorescent undergoes a light-catalyzed radical reaction with trichloroethanol (TCE), that installs an alpha hydroxy ketone on the tryptophan indole ring, extending the conjugation and shifting the intrinsic fluorescence of tryptophan 100 nm to the red end of the spectrum.

[0199] To illustrate further how amino acid types can be specifically and fluorogenically labelled, a table of fluorescent dyes and reaction approaches is presented below for use with the invention, from which an appropriate label and reaction strategy can be chosen for each reaction type. Table 3: Fluorogenic labelling for each R-group of each amino acid type Amino acid type R-group labelled Fluorogenic Labelling Alanine (A)MethylPalladium catalysed C(sp 3 )-H 3 bond activation, Pd(OAc) 2 with 1-ethynyl-4-iodobenzene, to install alkyne followed by Cu(I) catalyzed azide-alkyne cycloaddition(CuAAC) "click-chemistry" with 3-azido-2H-chromen-2-one.λex = 365 nm, λem = 478 nmArginine (R)aliphatic guanidino group; partial primary amine character and / or an equal primary amine characterDopachrome, pH 10.5, 20 mM dopachromeλex = 380 nm, λem = 480 nmAsparagine (N)β-carboxamide4-amino-3-formylphenyl nitrateλex = 350 nm, λem = 450 nmAspartic acid (D)β-carboxylic acid4-(diethylamino)-2-(pyridin-2-ylmethoxy)benzaldehyde appended BODIPY based probeλex = 500 nm, λem = 510 nmCysteine (C)Reduced thiol4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) at pH 10.5ORo-maleimide BODIPYλex = 500 nm, λem = 510 nmORethyl (Z)-2-(6-(ethyl((3-(trifluoromethyl)phenyl)selanyl)amino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl)benzoateCysteine (C)Reduced thiol4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) at pH 10.5ORo-maleimide BODIPYλex = 500 nm, λem = 510 nmORethyl (Z)-2-(6-(ethyl((3-(trifluoromethyl)phenyl)selanyl)amino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl)benzoate after tris(2-carboxyethyl)phosphine (TCEP)Glutamic acid (E)γ-carboxylic acid4-(diethylamino)-2-(pyridin-2-ylmethoxy)benzaldehyde appended BODIPY based probeλex = 500 nm, λem = 510 nmGlutamine (Q)γ-carboxamide4-amino-3-formylphenyl nitrateλex = 500 nm, λem = 600 nmGlycine (G)hydrogenC-H bond functionalization alpha to carbonyl via reaction with H-alkynyl-Phe in the presence of CuBr (10 mol-%) and 1 equivalent (eq.) of tBuOOH in DCM, followed by CuAAc with 3-azido-7-methoxy-2H-chromen-2-oneλex = 365 nm, λem = 420-449 nmhistidine Histidine (H)Imidazole2-butyl-6-(4-((6-(((2-ethoxyethyl)amino)methyl)pyridin-2-yl)methyl)piperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione-Cu 2+< Isoleucine (I)sec-butylBlue light meditated Hoffman-Loffler-Freytag reaction for δ-C-H functionalization of isoeleucine: Reaction with acetic hypobromous anhydride catalyzed by blue LED to install a Br group, followed by SN2 reaction with KN 3 to install azide group, then CuAAc with 4-((7-ethynyl-2-oxo-2H-chromen-4-yl)methoxy)-4-oxobutanoic acidLeucine (L)IsobutylBlue light meditated Hoffman-Loffler-Freytag reaction for δ-C-H functionalization of isoeleucine: Reaction with acetic hypobromous anhydride catalyzed by blue LED to install a Br group, followed by SN2 reaction with KN 3 to install azide group, then CuAAc with 4-((7-ethynyl-2-oxo-2H-chromen-4-yl)methoxy)-4-oxobutanoic acidLysine (K)ε-amino groupOrtho-phthalaldehyde (OPA) in the presence of β-mercaptoethanol (BME)Methionine (M)S-methyl thioetherReaction with alkyne bearing methionine-selective iodonium salt, followed by click chemistry with CalFlour dye λex = 488 nm, λem = 520 nmPhenylalanine (F)BenzylPalladium catalysed alkynylation reaction with (bromoethynyl)triisopropylsilane (10 mol % Pd(OAc)2 with 2 equivalents of K2CO3 as a base, and 0.2 equiv of PivOH as an additive), followed by CuAAc with 3-azido-7-hydroxy-2H-chromen-2-oneλex = 365 nm, λem = 490-499 nmProline (P)pyrrolidineamphiphilic dipolar Schiff baseZn II< complexesSserine (S)HydroxymethylSelective conversion to azide with TT / n-Bu4NN3 or Ph3P:2,3-dichloro-5,6- dicyanobenzoquinone (DDQ):n-Bu4NN3 followed by reaction with FI-DIBOThreonine (T)HydroxylSelective conversion to azide with TT / n-Bu4NN3 or Ph3P:2,3-dichloro-5,6- dicyanobenzoquinone (DDQ):n-Bu4NN3 followed by reaction with FI-DIBOTryptophan (W)IndoleTrichloroethanol (TCE)Tyrosine (Y)PhenolTrichloroethanol (TCE)ORInstallation of aryl groups ortho to the tyrosine hydroxyl groups using [RhCl(PPh 3 ) 3 ] (5 mol-%), R 2 P(OAr) (20 mol-%)Ar-Br (1.5 eq.), CsCO 3 (2 eq.)Ar = aryl, R = t-butyl, ArValine (V)IsopropylInstallation of quaternary azide group on valine side chain using [Ru(bpy)3]Cl2 (0.1 mol-%) catalyst and 2 eq. of 1-azido-1I3-benzo[d][1,2]iodaoxol-3(1H)-one catalysed by visible light, followed by fluorogenic CuAAC reaction with 4-((7-ethynyl-2-oxo-2H-chromen-4-yl)methoxy)-4-oxobutanoic acidPyrrolysine (O)pyrrol (N,2,3-trimethyl-3,4-dihydro-2H-pyrrole-2-carboxamide)Diels Alder reaction with an azaphthalimideSelenocysteine (U)ethylselenolABD-F, at pH 7

[0200] Strategies for the labelling of aliphatic amino acids exploit the state-of-the-art area of C-H bond functionalization (DOI: 10.1002 / ejoc.201800896). In alternative embodiments, a protease with cleavage specificity for an aliphatic (A, I, L, F or V) amino acid at the P1 or P1' position can be used to cut the protein sequence whenever the amino acid type of interest occurs. That generates a new protein N-terminus wherever the protein sequence has been cut. This can easily be modelled as the cleavage specificity for proteases is known. The protein N-terminus can react using a fluorogenic dye specific for the N-terminus such as an NHS-ester. In this way, a fluorogenic dye specific for the N-terminus reacts exclusively when the N-terminus is adjacent to the amino acid type of interest, hence, the concentration of an aliphatic, e.g. valine (V), amino acid type in the sample is measured based on the concentration of N-termini generated when the protease cleaves at the V position (and the signal of the label reports on the amino acid concentration of the V amino acid type). For example, human neutrophil elastase cleaves at valine amino acids. The number of V amino acids for the protein of interest is adjusted to add the number of N-termini already present within the protein of interest (based on the number of protein chains), and this is used as input to set of parametric equations 1. In some embodiments, the protease also cleaves, generating signal due to its own valine amino acids, but this is incorporated into the background fluorescence intensity measurement.

[0201] Preferably, the R-groups labelled for the amino acid types are the R-groups for the amino acid types; these include labelling of R-groups containing a glycoside specific for R-groups containing a glycoside and comprises Selective conversion to azide with TT / n-Bu4NN3 or Ph3P:2,3-dichloro-5,6-dicyanobenzoquinone (DDQ):n-Bu4NN3 followed by reaction with FI-DIBO. Labelling of R-groups containing a fatty acid is specific for R-groups containing a fatty acid comprises labelling with Dipolar 3-methoxychromones, allowing detection of all lipidated amino acid types. Labelling of R-groups containing a phosphate comprises activation with carbonyldiimidazole to provide a leaving group, followed by reaction with a cysteine BODIPY dye, and is specific for R-groups containing a phosphate, allowing detection of all amino acid types modified with a phosphate.

[0202] In some embodiments, the modified amino acids of an amino acid type are labelled differently to the unmodified amino acids of the amino acid type. For example, for the amino acid type cysteine, the labelling reaction for the unmodified amino acids (e.g. R-group of) C R is different to the labelling reaction for the combination of the modified and unmodified amino acids (e.g. R-group of, once the modified amino acids have been reduced). In the C R labelling reaction, only reduced cysteine amino acids are available for modification. The disulphide bonded cysteine amino acids are not available for modification. To label both modified and unmodified amino acids of cysteine, both the reduced cysteine amino acids and disulphide bonded cysteine amino acids need to be labelled. One way to achieve this is to reduce the disulphide bonded cysteine amino acids to reduced cysteine amino acids before being labelled. Preferably, the disulphide bonded cysteine amino acids are reduced with TCEP before labelling all of the reduced cysteine, that includes the oxidsied cysteine which has newly been reduced i.e. the combination of C D and C R , after C D has been reduced, with ABD-F. This reduction of the oxidized cysteine amino acids allows both the disulphide bonded and the reduced cysteine amino acids in the sample to be labelled, i.e. the combination of C D and C R . By fractionating the sample into multiple fractions, it is possible to measure C D , C R and / or the combination of C D and C R T amino acids within the sample. In some embodiments, the sample is separated into two fractions. In one fraction, C R is labelled. In the second fraction, the combination of C D and C R is labelled. The number of C D labelled is equal to the number of the combination of C D and C R labelled per protein minus the number of C R labelled.

[0203] The skilled person would appreciate that although all or a proportion of amino acids within a peptide are exposed to the solvent and available for a labelling reaction, this may not be the case for a folded protein. In some embodiments, a sample is denatured prior to or during the labelling reaction. Methods for denaturing a protein are known in the art. In some embodiments, this is achieved via adding a miscible organic solvent such as dimethyl sulfoxide, methanol, acetonitrile, ethanol, or isopropanol. In some embodiments, this is achieved via changing the buffer conditions to low or high pH such as pH 2, pH 3, pH 4, pH5, pH 7.5, pH 8.5, pH 9, pH 10, or pH 10.5. In some embodiments, this is achieved by heating the solution to 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, or 100 °C. In some embodiments, this is achieved by reducing protein disulphide bonds with TCEP, β-mercapto ethanol, DTBA, or DTT. In some embodiments, this is achieved by adding a denaturing agent such as urea, guanidinium chloride, or guanidinium thiocyanate. In some embodiments, this is achieved by adding a surfactant such as sodium dodecyl sulfate (SDS), dodecyltrimethylammonium bromide (DTAB), cetyltrimethylammonium bromide (CTAB), phosphatidylcholine, Triton X-100, Triton X-114, CHAPS, NP-40, sodium 1-undecanesulfonate (SUS) sodium dodecylbenzenesulfonate (SDBS), sodium deoxycholate (DOC), sodium stearate, 4-(5-dodecyl)benzenesulfonate, dioctyl sodium sulfosuccinate, alkyl ether phosphates, benzalkaonium chloride (BAC), and perfluorooctanesulfonate (PFOS). In some embodiments, denaturing proteins contained within a sample is achieved during the labeling reaction. In some embodiments, the labelling reactions are performed in the presence of the additives listed herein. In some embodiments, denaturing polypeptides contained within a sample is achieved by reducing polypeptide disulphide bonds and adding a surfactant. In some embodiments, denaturing proteins contained within a sample is achieved by reducing protein disulphide bonds and adding a surfactant. In some embodiments, denaturing polypeptides contained within a sample is achieved by reducing polypeptide disulphide bonds, adding a surfactant and changing the buffer conditions to high or low pH. In some embodiments, denaturing proteins contained within a sample is achieved by reducing protein disulphide bonds, adding a surfactant and changing the buffer conditions to high or low pH. In some embodiments, denaturing proteins contained within a sample is achieved by reducing protein disulphide bonds with TCEP and adding the surfactant SDS. In some embodiments, denaturing proteins, peptides, oligopeptides, polypeptides, and / or protein complexes which comprise a subproteome or a proteome contained within a sample is achieved by reducing protein, peptide, oligopeptide, polypeptide, and / or protein complex disulphide bonds and adding a surfactant. In some embodiments, denaturing proteins, peptides, oligopeptides, polypeptides, and / or protein complexes which comprise a subproteome or a proteome contained within a sample is achieved by reducing by reducing protein, peptide, oligopeptide, polypeptide, and / or protein complex disulphide bonds, adding a surfactant and changing the buffer conditions to high or low pH. In some embodiments, denaturing proteins, peptides, oligopeptides, polypeptides, and / or protein complexes which comprise a subproteome or a proteome contained within a sample is achieved by reducing protein, peptide, oligopeptide, polypeptide, and / or protein complex disulphide bonds, adding a surfactant and changing the buffer conditions to high or low pH. In some embodiments, denaturing proteins, peptides, oligopeptides, polypeptides, and / or protein complexes which comprise a subproteome or a proteome contained within a sample is achieved by reducing protein, peptide, oligopeptide, polypeptide, and / or protein complex disulphide bonds with TCEP and adding the surfactant SDS. For example, the labelling reactions are performed at pH 10 in the presence of 4% w / v SDS and 18 mM β-mercaptoethanol. As another example, the labelling reactions are performed at pH 10.5 in the presence of 4% SDS and 10 mM TCEP. In some embodiments, there are multiple steps to the labelling reaction. In some embodiments, the first step makes the R-group of an unmodified or modified amino acid type reactive for labelling, and proceeds under a set of conditions appropriate for that reaction. Then the second step of the labelling reacts the now reactive R-group under the denaturing conditions described for the labelling of all amino acids of the amino acid type. For example, labelling of the the combination of C D and C R amino acid type first involves reduction of the C D amino acid subtype with 10 mM TCEP to expose reactive thiols, and proceeds at pH 7 within 45 minutes. Then, the exposed thiols are reacted with ABD-F at pH 10.5 in the presence of 4% SDS. As another example, the first step of labelling phenylalanine is making the R-group reactive for labelling, and involves a palladium catalysed alkynylation reaction with (bromoethynyl)triisopropylsilane in the presence of 10 µM Pd(OAc) 2 with 10 mM K 2 CO 3 as a base, and 1 mM PivOH as an additive in water. This installs an alkyne group onto the phenylalanine ring, which is specifically reactive for azide groups. The next step of the labelling reaction involves CuAAc with 3-azido-7-hydroxy-2H-chromen-2-one in 75% H2O / 25% BuOH in the presence of 5 µM CuSO4 and 25 µM Na ascorbate.

[0204] In some embodiments, the amino acids of (e.g. R-group), the combination of C D and C R are fluorogenically labelled with ABD-F after reduction with TCEP, and denaturation with sodium dodecyl sulfate (SDS) in a buffer. In some embodiments, the TCEP buffer is HEPES buffer and the ABD-F / SDS buffer is sodium carbonate buffer. In some embodiments, the amino acids of the combination of C D and C R are fluorogenically labelled with 2-10 mM ABD-F, 2-10% SDS and 50-500 mM sodium carbonate buffer. In some embodiments, the amino acids of the combination of C D and are fluorogenically labelled with 5 mM ABD-F, 4% (SDS) in 80 mM sodium carbonate buffer. In some embodiments, the timing of the TCEP reaction is from about 20 to about 40 minutes, preferably 30 minutes. In some embodiments, the timing of the ABD-F reaction is from about 5 to about 55 minutes, preferably about 45 minutes.

[0205] In some embodiments, the amino acids, (e.g. R-group) of reduced cysteine (C R ) are fluorogenically labelled with ABD-F after denaturation with SDS in a buffer. In some embodiments, the buffer is a sodium carbonate buffer. In some embodiments, the amino acids of C R are fluorogenically labelled with 2-10 mM ABD-F, 2-10% SDS and 50-500 mM sodium carbonate buffer. In some embodiments, the amino acids of C R are fluorogenically labelled with 5mM ABD-F, 4% SDS in 80 mM sodium carbonate buffer.

[0206] In some embodiments, the unmodified amino acids of the amino acid type, (e.g. R-group) of lysine (K) are fluorogenically labelled with OPA, β-mercaptoethanol (BME) and SDS in a buffer. In some embodiments, the buffer is a sodium carbonate buffer. In some embodiments, the amino acids are fluorogenically labelled with 10-20 mg OPA + 5-10 mL carbonate buffer + 10-20 µL BME + 1-5 mL 20% SDS. In some embodiments, the amino acids are fluorogenically labelled with 12 mM ortho-phthalaldehyde (OPA), 18 mM beta-mercaptoethanol (BME), 4% SDS in 200 mM sodium carbonate buffer. The dye molecule, OPA, is a dialdehyde. The lysine primary amine attacks one aldehyde and water is lost. This results in the formation of an imine, specifically a Schiff base. The thiol nucleophile presented by BME attacks this Schiff base, such that the amine is again available for a ring-closing attack on the other pendant aldehyde. BME participates in the reaction to create the fluorophore, however, other thiols can be used instead of BME. Water is lost, and conjugation is extended into the newly formed ring, resulting in generation of fluorescence into the visible region of the spectrum.

[0207] In some embodiments, the combination of the modified and unmodified amino acids of the amino acid type (e.g. R-group) of tryptophan (W) and the combination of the modified and unmodified amino acid type (e.g. R-group) of tyrosine (Y) are fluorogenically labelled with TCE, after reduction with TCEP, and denaturation with sodium dodecyl sulfate (SDS) in a buffer. TCEP reduces the disulphide bonds within the protein such that, together with SDS denaturation, all tryptophan (W) and tyrosine (Y) amino acids are available for reaction. In some embodiments, the buffer is HEPES buffer. In some embodiments, the excited state of W amino acids, Y amino acids, or W and Y amino acids undergoes a radical reaction with TCE. In some embodiments, the reaction is photo-catalyzed with UV light of a wavelength absorbed by W amino acids, Y amino acids, or W and Y amino acids. In some embodiments, the amino acid type of W is fluorogenically labelled with 0.01-5 M TCE, 2-50 mM TCEP and 2-20% SDS in 1-10 mM HEPES catalyzed by UV light with wavelengths of 260-310 nm. In some embodiments, the amino acid type of W is fluorogenically labelled in 0.2 M TCE, 10 mM TCEP and 4% SDS in 5 mM HEPES catalyzed by UV light with wavelengths of 295-305 nm. In some embodiments, the amino acid type of Y is fluorogenically labelled with 0.01-5 M TCE, 2-50 mM TCEP and 2-20% SDS in 1-10 mM HEPES catalyzed by UV light with wavelengths of 260-310 nm. In some embodiments, the amino acid type of Y is fluorogenically labelled in 0.2 M TCE, 10 mM TCEP and 4% SDS in 5 mM HEPES catalyzed by UV light with wavelengths of 285-295 nm.

[0208] In some embodiments, when labelling the amino acid type (e.g. R-group) cysteine (C), wherein both the modified and the unmodified amino acids of cysteine are labelled, the oxidized (disulphide bonded) cysteines are reduced via a reducing agent (TCEP), before the cysteine thiol contained within the cysteine R-group acts as a nucleophile in an electrophilic addition / elimination reaction on the dye ABD-F. This results in loss of a fluorine quenching group, such that the fluorescence of the dye is no longer quenched. When labelling the amino acid type lysine (K), the lysine primary amine contained within the R-groups attacks one of the aldehydes contained within OPA. This forms an imine which is attacked by a thiol (BME) added to the reaction, releasing a primary amine for attack on the remaining pendant aldehyde. This closes a second ring and extends the aromatic conjugation bringing the fluorescence into the visible region.

[0209] The labelling reaction for the amino acid types tryptophan and tyrosine is a photo-catalyzed radical reaction. Tryptophan can be labelled with 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA) or chloroform, as well as other di / tri halogenated compounds. Radicals from the tryptophan R-group and TCE combine, and a hydrogen atom is lost resulting in the addition of a dihalo compound to the indole ring. This is unstable and attacked by a water molecule, then hydrochloric acid is lost, resulting in the addition of an alpha hydroxy ketone to the indole ring which shifts the intrinsic fluorescence of tryptophan about 100 nm to the right and into the visible region. The labelling reaction for tyrosine reaction is also a photo-catalyzed radical reaction with TCE which shifts the intrinsic fluorescence of tyrosine about 100 nm to the right and into the visible region. The phenol R-group of tyrosine combines with TCE resulting in the addition of an alpha hydroxy ketone to the ring via the same mechanism as in the tryptophan labelling reaction.

[0210] It was discovered that labelling an already intrinsically fluorescent amino acid type can enable quantitative detection not possible for the unlabelled amino acid type. For example, tryptophan and tyrosine amino acids are intrinsically fluorescent, so labelling intrinsically fluorescent amino acids with a fluorescent label is counterintuitive. It is appreciated by those skilled in the art that intrinsic fluorescence for tryptophan and tyrosine amino acid types is highly dependent on the local environment surrounding these residues within the 3D-protein structure, therefore intrinsic fluorescence from the tryptophan and tyrosine amino acid types cannot reveal the content of tryptophan and tyrosine residues within protein sequences. However, when these amino acid types are labelled, the environmental sensitivity of their fluorescence disappears, and the fluorescence from the labelled tryptophan or tyrosine does reveal the number or concentration of tryptophan or tyrosine amino acid types within the sample. For example, a disappearance of environmental sensitivity of the fluorescence is observed for tryptophan and tyrosine amino acid types labelled with trichloroethanol (TCE). This unexpected result could be due to a change in the electronic properties of tryptophan or tyrosine whose indole or phenol ring conjugation has been extended via the addition of an alpha hydroxy ketone, making the fluorophore less sensitive to the polarity of the local environment.

[0211] Alternatively, the fluorescent label includes a fluorescent protein or conjugated antibody. Preferably, the fluorescent protein is selected from the group consisting of: CFP, GFP (emGFP), RFP (tagRFP), GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, mCerulean, mCerulean3 mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midoriishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 ("RFP"), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum or mRaspberry. In some embodiments, conjugated antibodies specific for post-translational modifications can be used within the methods of the invention. In some embodiments, the conjugated antibody is labelled with one of the fluorescent labels or fluorophores provided herein. Preferably, the conjugated antibody is a monoclonal antibody derived traditionally or synthetically, and is selected from the group including IgG, IgM, IgA, IgE or nanobodies. Preferably, the antibody is labelled with one or a fluorogenic dye, fluorescent label, or fluorophore per antibody. In some embodiments, the conjugated antibody is selective for a post-translational modification including: N-acetylation, methylation, deimination to citrulline, deamidation to aspartic acid or isoaspartic acid, N-linked glycosylation, isomerization to isoaspartic acid, disulfide-bond formation, oxidation to sulfenic, sulfinic or sulfonic acid, palmitoylation, N, acetylation (N-terminus), S-nitrosylation, cyclization to Pyroglutamic acid (N-terminus), gamma-carboxylation, deamidation to glutamic acid, isopeptide bond formation, N-Myristoylation (N-terminus), Phosphorylation, Ubiquitination, SUMOylation, isopeptide bond formation to a glutamine, hydroxylation, N-linked Ubiquitination, oxidation to sulfoxide or sulfone, Hydroxylation, O-linked glycosylation, mono- or di-oxidation, formation of Kynurenine, or sulfation.

[0212] Alternatively, the label is a tandem mass tag (TMT). Preferably, the tandem mass tags are TMTzero, TMTduplex, TMTsimplex, TMT 10-plex, TMTpro and TMTpro Zero. Alternatively, the label is a stable isotope label (i.e. isotopic labelling). In some embodiments, the stable isotope label is a non-radioactive isotope. In some embodiments, the non-radioactive isotope label is 2H, 13C, and / or 15N. In some embodiments, the labelling strategies are used in combination. For example, each amino acid type can be labelled with chemically (e.g. with a fluorogenic dye) and then labelled with an antibody. For example, three or more amino acid types are labelled chemically, and then a post-translational modification specific antibody is used, for example, to detect phosphorylation of amino acids of a different amino acid type.

[0213] It would be appreciated by the person skilled in the art, that the labelling reactions encompassed as part of the invention, can be performed without, or following a separation step to isolate the protein component of the sample, or a particular protein of interest in the sample. For example, a separation step such as extraction, precipitation and differentiation solubilization, centrifugation, ultracentrifugation, sonication, size exclusion chromatography, separation based on charge or hydrophobicity (examples include hydrophobic interaction chromatography, ion exchange chromatography, free-flow electrophoresis, capillary electrophoresis), affinity chromatography such as immunoaffinity chromatography and high-performance liquid chromatography (HPLC), or other methods known within the technical field can be used.

[0214] In some embodiments, the proteins within the sample are concentrated once isolated. This can involve, but is not limited to, lyophilization or ultrafiltration. In some embodiments, one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes within a sample are concentrated once isolated.

[0215] In some embodiments, the label is a fluorescent label and three or more amino acid types are labelled with the same fluorescent label under the same conditions, but the labelling reactions proceed at different rates. Therefore, measuring the time-resolved signal of the label at a certain time reveals the signal of the label from exclusively one labelled amino acid type, while measuring the time-res...

Claims

1. An in vitro method of identifying the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest within a sample, the method comprising: a) Labelling three or more amino acid types within the sample, wherein an amino acid type is defined by the R-group of the amino acid; b) measuring the label of each labelled amino acid type in the sample; c) optionally calculating the amino acid concentration of each labelled amino acid type from the measured label; d) optionally calculating the number of amino acids of each labelled amino acid type; and e) identifying the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest in the sample by comparing the measured label and / or amino acid concentration of each labelled amino acid type in the sample to the known label values and / or amino acid concentrations of the same three or more amino acid types that have been labelled in the sample of each of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest at one or more concentrations, or comparing the number of amino acids of each labelled amino acid type in the sample to the known number of amino acids of the same three or more amino acid types that have been labelled in the sample in the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest using an n-dimensional space corresponding to the n labelled amino acid types.

2. The method of claim 1, wherein the three or more amino acid types are selected from the group consisting of: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), pyrrolysine (O), selenocysteine (U), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V) or synthetic amino acids and any combination thereof, wherein the R-group of an amino acid type is labelled.

3. The method of claim 1 or 2, wherein the three or more amino acid types comprise modified amino acids of an amino acid type that are post translationally modified amino acids of the amino acid type and wherein the modified amino acids of an amino acid type are labelled independently to unmodified amino acids of that amino acid type.

4. The method of any one of claims 1-3, wherein the label is a fluorescent label, wherein the fluorescent label is a fluorescent dye, fluorogenic dye, and / or molecule which becomes fluorescent upon reaction with an amino acid type.

5. The method of any one of claims 1-4, wherein the amino acid concentration of each labelled amino acid type in the sample is calculated from the measured label and the amino acid concentration is calculated from the measured label using a calibration curve or standard which converts between the measured label of the sample and the amino acid concentration of that amino acid type in the sample using the measured labels of one or more known amino acid concentrations of one or more proteins or amino acids, or, wherein the number of amino acids of each labelled amino acid type in the sample is calculated, and the number of amino acids of each labelled amino acid type in the sample is calculated by dividing the amino acid concentration of that amino acid type in the sample by the total molar concentration of the sample.

6. The method of any one of claims 1-5, wherein information indicating the known label values and / or amino acid concentrations, and / or number of amino acids of the same three or more amino acid types as the amino acid types that have been labelled in the sample which identifies the presence and / or concentration of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is obtained from a database, or is a reference, wherein the reference provides the known label values and / or amino acid concentrations of the same three or more amino acid types as the amino acid types that have been labelled in the sample of each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest as a set of parametric equations or a vector function depending on the common parameter of concentration, and / or, wherein the reference provides the number of amino acids of the same three or more amino acid types as the amino acid types that have been labelled in the sample of each protein, peptide, oligopeptide, polypeptide, protein complex, subproteome, or proteome of interest.

7. The method of any one of claims 1-6, wherein the known label values and / or amino acid concentrations, and / or number of amino acids of the same three or more amino acid types as the amino acid types that have been labelled in the sample at one or more concentrations of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest is calculated from the amino acid sequence or sequences of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest and / or experimental information about post-translation modifications of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest.

8. The method of claim 1, wherein step e) comprises comparing the measured label of each labelled amino acid type of the three or more labelled amino acid types in the sample to the known label values of each of the same three or more amino acid types as the amino acid types that have been labelled in the sample of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest at one or more concentrations, or, comparing the amino acid concentration of each labelled amino acid type in the sample to the amino acid concentrations of the same three or more amino acid types as the amino acid types that have been labelled in the sample of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest at one or more concentrations, or, comparing the number of amino acids of each labelled amino acid type of the three or more labelled amino acid types in the sample to the number of amino acids of the same three or more amino acid types as the amino acid types that have been labelled in the sample of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest.

9. The method of any one of the preceding claims, wherein the known label values and / or amino acid concentrations and / or number of amino acids of the same three or more amino acid types as the amino acid types that have been labelled in the sample in a proteome or subproteome of interest is a weighted mean of the known label values, amino acid concentrations or number of amino acids of each amino acid type of all amino acid sequences contained within the proteome or subproteome of interest.

10. The method of any one of the preceding claims, wherein the proteome and / or subproteome of interest is the HIV proteome and / or a subproteome thereof; and / or theSARS-CoV-2 proteome and / or a subproteome thereof; and / or a zoonotic proteome and / or a subproteome thereof; and / or a host response to an infection proteome and / or subproteome thereof; and / or a cancer proteome and / or subproteome thereof.

11. The method of any one of claims 1-10, where the method comprises identifying the presence and / or concentration and / or amount of one or more cancer proteomes and / or cancer subproteomes of interest within a sample.

12. The method of any one of claims 1-11, wherein step (e) comprises comparing the measured label and / or amino acid concentration of each labelled amino acid type in the sample to the known label values and / or amino acid concentrations of the same three or more amino acids types that have been labelled in the sample of each of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest at one or more concentrations using a machine learning classifier.

13. An in vitro method of detecting cancer, the method comprising: a) Labelling three or more amino acid types within a sample, wherein an amino acid type is defined by the R-group of the amino acid; b) measuring the label of each labelled amino acid type in the sample; c) optionally calculating the amino acid concentration of each labelled amino acid type from the measured label; d) optionally calculating the number of amino acids of each labelled amino acid type; and e) identifying the presence and / or concentration and / or amount of one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest related to cancer in the sample by comparing the measured label and / or amino acid concentration of each labelled amino acid type in the sample to the known label values and / or amino acid concentrations of the same three or more amino acid types that have been labelled in the sample of each of the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest related to cancer at one or more concentrations, and / or comparing the number of amino acids of each labelled amino acid type in the sample to the known number of amino acids of the same three or more amino acid types that have been labelled in the sample in the one or more proteins, peptides, oligopeptides, polypeptides, protein complexes, subproteomes, or proteomes of interest related to cancer using an n-dimensional space corresponding to the n labelled amino acid types.