Quality Analysis

Abstract

The present invention provides isotopic mass tags for labeling peptides and other biomolecules, and a method for detecting analytes by identifying one or more of the mass tags or combinations thereof by mass spectrometry.

Description

[Technical Field]

[0001] (Technical field) This disclosure relates to a method for assaying target analytes, particularly biomolecules such as nucleic acids and proteins, by mass spectrometry. Specifically, this disclosure relates to a multiplexed mass spectrometry method using a set of mass labels. Within a given set, the mass labels are isotopologs, meaning they have the same structural formula and differ only in their isotopic composition. As is common in the literature, such isotopolog labels will be referred to as isotopic labels in this disclosure. [Background technology]

[0002] (Brief explanation) The study of biological systems, and especially the understanding of human diseases, depends on the ability to detect changes in biological systems caused by or in response to disease. Such changes provide diagnostic tools and insights into targets for therapeutic compounds such as vaccines and pharmaceuticals. A wide range of biomolecules, including nucleic acids, proteins, steroids, sugars, and lipids, need to be quantitatively measured to understand disease processes. In this context, the ability to quantitatively detect such biomolecules using mass spectrometers has led to considerable progress in their study and its application to human and veterinary diseases. Similar progress has been made in environmental analysis and monitoring, as well as in food and beverage production. In particular, the use of stable isotopes to provide reference substances for the quantitative analysis of synthetics has been developed in isotopic dilution mass spectrometry for the monitoring of all classes of biomolecules. However, these methods have traditionally required readily available synthetic standards, which is not always possible.

[0003] In recent years, various chemical mass tags with heavy isotope substitution have been developed to further improve the quantitative analysis of biomolecules by mass spectrometry. Depending on the tag design, the members of the tag set are either isotopic, having the same chemical structure but different absolute masses, or isobaric (i.e., often even more isotopomeric), having both the same structure and the same absolute mass but different exact arrangements of heavy isotopes. Isotopic tags are typically used for quantification in MS mode, while isobaric tags must be fragmented in MS / MS mode to release a reporter fragment with a unique mass.

[0004] An early example of isotopic mass tagging was the isotope-coded affinity tag ("ICAT") (Gygi, SP et al., (1999) Nature Biotechnology, 17, 994-999). The ICAT reagent is a pair of mass tags in which one tag (heavy tag) has differential incorporation of heavy isotopes, and the other tag (light tag) has no substitutions. Two samples are labeled with either the heavy or light tag, then mixed and analyzed by LC-MS. The peptides present in both samples give a pair of precursor ions with different masses proportional to the number of heavy isotope atom substitutions.

[0005] Furthermore, the ICAT method is a useful example of a “sampling” method that reconciles the need to handle small populations of peptides to reduce the complexity of the resulting mass spectrum while retaining enough information about the original sample to identify its components. The “isotope-coding affinity tag” used in the ICAT method contains a pair of biotin linker isotopes that are reactive to thiols for capturing cysteine-containing peptides. Typically, 90–95% of proteins in the proteome will have at least one cysteine-containing peptide, and usually there is about one cysteine-containing peptide per 10 peptides overall. Therefore, the analysis of cysteine-containing peptides greatly reduces the complexity of the sample without losing important information about the sample. Accordingly, in the ICAT method, a sample of protein from one source is reacted with a “light” isotope biotin linker, and a sample of protein from a second source is reacted with a “heavy” isotope biotin linker that is typically 4–8 Daltons ("Da") heavier than the light isotope. The two samples are then pooled and cleaved with endopeptidase. The biotinylated cysteine-containing peptides can then be isolated on avidinated beads for subsequent mass spectrometry analysis. The two samples are quantitatively comparable: the corresponding peptide pairs act as mutual standards, allowing their ratios to be quantified. While the ICAT sampling method still accurately represents the source sample and produces a less complex peptide mixture than MudPIT, numerous peptides are still isolated, and their analysis by LC-MS / MS generates complex spectra. Using two ICAT tags doubles the number of peptide ions in the mass spectrum compared to unlabeled analysis. Furthermore, isotopic tags such as IPCL, mTRAQ, and dimethyl labeling enable sample multiplexing in MS1.Such isotopic labeling increases the number of precursor ions as a function of the multiplexing level; that is, a quadruple set of tags generates four times the number of precursors, thus increasing the time required for MS1 multiplexed data-dependent acquisition (DDA) analysis (see Mertins, P. et al., (2012) Molecular & Cellular Proteomics 11), which can significantly impact the level of protein coverage. In practical terms, this has limited the set of isotopic tags to a maximum of four members.

[0006] However, more recently, techniques for multiplexed protein analysis using isotopic labeling have been developed using data-independent acquisition (DIA) methods (e.g., plexDIA or mDIA) that exponentially increase the processing capacity with the number of labels without reducing proteome coverage or quantitative accuracy (see Derks, Jason et al., (2023) Nature biotechnology 41.1: 50-59; Thielert, Marvin et al., bioRxiv preprint doi: https: / / doi.org / 10.1101 / 2022.12.02.518917). [Overview of the project]

[0007] (Brief explanation) Considering the drawbacks of existing mass labels, there is still a need for a set of isotopic tags for labeling peptides and other biomolecules that maintain the retention time in the same chromatography and provide unique mass-to-charge ratios for each version of the same peptide sequence while enabling a higher level of sample multiplexing. This is because relative quantification is performed at the MS1 level of tandem mass spectrometry, and the identification of sets of peptides having similar mass-to-charge ratios can be achieved within the scope of the DIA method described herein using a window of defined width in the range from 100 Da to less than 10 Da using the isotopic mass labels described herein. To maximize the multiplexing level while maintaining sufficient resolution, each isotopic tag should be separated from all other tags in the set by at least a difference of 0.003 Da, a difference of 0.006 Da, and in some cases at least a difference of 1 Da.

[0008] In a first general aspect, the present disclosure is a set of two or more isotopic mass labels, each label having the formula:

Chemical formula

Chemical formula

[0009] In another aspect, X has the general formula:

Chemical formula

[0010] In some embodiments, the present disclosure relates to a set of two or more isotopic mass labels, Each mass label is given by the formula: [ka] (In the formula, X is a coding region having precise mass, L is a bond that can be arbitrarily severed by collision in a mass spectrometer, M is a mass modification region, and A is the analyte.) including and The present invention relates to a set of isotopic mass labels, wherein each mass label in the set has a different integer mass. In some embodiments, X is given by the general formula: [ka] Includes.

[0011] In some embodiments, the set of two or more isotopic mass labels has the following structure: [ka] (In the formula: Each R1 is independently H, a substituted or unsubstituted linear or branched C1-C6 alkyl group, a substituted or unsubstituted aliphatic cyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heterocyclic group, or an amino acid side chain. a is an integer between 0 and 10. b is at least 1, and c is at least 1. It includes one or more mass labels having the following properties.

[0012] In some embodiments, the set of two or more isotopic mass labels has the following structure: [ka] (In the formula: Each R1 is independently H, a substituted or unsubstituted linear or branched C1-C6 alkyl group, a substituted or unsubstituted aliphatic cyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heterocyclic group, or an amino acid side chain. a is an integer between 0 and 10. b is at least 1, c is at least 1, and * indicates an isotope mass regulation site, and oxygen is present. 18 Is it O, or is it carbon? 13 Is it C, or nitrogen? 15 N, or hydrogen, 2 (This represents H, and may contain one or more * symbols.) It includes one or more mass labels having the following properties.

[0013] In some embodiments, the set of two or more isotopic mass labels has the following structure: [ka] (In the formula, * represents oxygen, 18 It is O, and carbon, 13 It is C, and nitrogen is 15 N is N, and hydrogen is 2 (This represents H, and may contain one or more * symbols.) Includes a mass label having a specific feature.

[0014] In some embodiments, the set of two or more isotopic mass labels has the following structure: [ka] Includes a mass label with n=6.

[0015] In some embodiments, the difference in precise mass between at least two of the mass markers is less than 100 millidaltons ("mDa"), less than 50 mDa, or less than 20 mDa. In some embodiments, the difference in precise mass between at least two of the mass markers is 2.5 mDa, 2.9 mDa, 6.3 mDa, 8.3 mDa, 9.3 mDa, or 10.2 mDa.

[0016] In another embodiment, the Disclosure relates to a mass spectrometry method comprising quantifying an analyte by mass spectrometry, wherein the mass label is a mass label derived from the set of mass labels according to the Disclosure, and the identification of the analyte is obtained from data-independent acquisition mass spectrometry.

[0017] In some embodiments, a mass spectrometry method includes: a) preparing a plurality of samples, each differentially labeled with an isotopic mass label or combination of isotopic mass labels, each selected from a set of two or more isotopic mass labels described herein; b) mixing the plurality of labeled samples to produce an analytical mixture containing labeled analytes; c) detecting the labeled analytes in a mass spectrometer; d) isolating one or more labeled analytes in the mass spectrometer based on one or more isolation windows of ≥1 Da, and then dissociating them to produce labeled analyte fragments; e) detecting the analyte fragments; and f) identifying the one or more labeled analytes based on the analyte fragments.

[0018] In some embodiments, the one or more isolation windows include isolation windows where ≤10Da, ≤20Da, ≤50Da, and / or ≤100Da.

[0019] In some embodiments, the dissociation in step d) is collision-induced dissociation in a mass spectrometer. In some embodiments, the dissociation is collision-induced dissociation in a mass spectrometer having a separation capability greater than 60,000 at a mass-to-charge ratio of 400, optionally, greater than 100,000 at a mass-to-charge ratio of 400, or greater than 250,000 at a mass-to-charge ratio of 400.

[0020] In some embodiments, the labeled analyte may be identified based on the MS2 spectrum of the labeled analyte fragment and quantified using the MS1 spectrum of the labeled analyte.

[0021] In some embodiments, each isotopic mass label is separated from all other tags in the set by a difference of at least 0.003 Da, 0.006 Da, or at least 1 Da. [Brief explanation of the drawing]

[0022] (Brief explanation of the drawing) [Figure 1] Figure 1 shows the reaction scheme for the synthesis of 3-tert-butoxycarbonylaminopropionic acid (Boc-βAla-OH).

[0023] [Figure 2] Figure 2 shows the reaction scheme for the synthesis of benzyl 3-aminopropionate hydrotosylate (H-βAla-OBn*pTosOH).

[0024] [Figure 3] Figure 3 shows the reaction scheme for the synthesis of 3-[3-(tert-butoxycarbonylamino)propanoylamino]benzyl propanoate (Boc-βAla-βAla-OBn).

[0025] [Figure 4] Figure 4 shows the reaction scheme for the synthesis of 3-(3-aminopropanoylamino)propanoate benzyl hydrochloride (H-βAla-βAla-OBn*HCl).

[0026] [Figure 5] Figure 5 shows the reaction scheme for the synthesis of 3-[3-[3-(tert-butoxycarbonylamino)propanoylamino]propanoylamino]benzyl propanoate (Boc-βAla-βAla-βAla-OBn).

[0027] [Figure 6] Figure 6 shows the reaction scheme for the synthesis of 3-[3-(3-aminopropanoylamino)propanoylamino]benzyl hydrochloride (H-βAla-βAla-βAla-OBn*HCl).

[0028] [Figure 7] Figure 7 shows the reaction scheme for the synthesis of 3-[3-[3-(diisobutylamino)propanoylamino]propanoylamino]benzylpropanoate (diisobutyl-βAla-βAla-βAla-OBn).

[0029] [Figure 8] Figure 8 shows the reaction scheme for the synthesis of 3-{3-[3-(diisobutylamino)propionylamino]propionylamino}propanoic acid (diisobutyl-βAla-βAla-βAla-OH).

[0030] [Figure 9] Figure 9 shows the reaction scheme for the synthesis of 3-[3-[3-(diisobutylamino)propanoylamino]propanoylamino]propanoic acid (2,5-dioxopyrrolidine-1-yl) (diisobutyl-βAla-βAla-βAla-OSu).

[0031] [Figure 10] Figure 10 shows the total mass spectral results for a set of six mass labels defined as Embodiment 1, exhibiting clean spectra with no additional signals of relevant intensity and a visible mass shift of 4 Da between each consecutive mass label in the set.

[0032] [Figure 11] Figure 11 shows the mass spectral results of a set of six mass labels defined as Embodiment 1, enlarged to emphasize the 400–500 Da range.

[0033] [Figure 12-17] Figures 12 to 17 show the total mass spectral results for each of the six mass labels defined as Embodiment 1.

[0034] [Figure 18] Figure 18 shows the relative elution profiles of trypsin peptides derived from bovine serum albumin labeled with isobaric TMT® reagent or isotopic tags according to this disclosure. Mobility of unlabeled peptides is shown for reference. The peptide peaks show good shape and do not exhibit front or rear shoulders.

[0035] [Figure 19-20] Figures 19–20 show MS / MS spectra of unlabeled (Figure 19) or isotopically labeled (Figure 20) bovine serum albumin-derived trypsin peptides. Sufficient structural ions are generated to enable reliable sequence assignment.

[0036] [Figure 21] Figure 21 shows the charge states and relative signal intensity distributions of complex proteomic samples (HeLa digests (Thermo Scientific)) analyzed either without labeling (top panel) or labeled with isotopic labeling as disclosed herein. The presence of tags increases the contribution of peptides with a 3+ charge state to the percentage and also increases their average intensity.

[0037] [Figure 22]Figure 22 shows that the peptide identification rate for isotopolog-tagged peptides charged to 3+ is considerably higher than that for other charge states, and is at a level similar to the typical identification rate in unlabeled proteomics experiments (comparative data are not shown).

[0038] [Figure 23] Figure 23 shows the labeling efficiency of each member of the six-plex pD-iB-βAla tag set in HeLa digestion samples as measured by mass spectrometry. As shown, all tags consistently exhibit high labeling efficiency.

[0039] [Figure 24-25] Figures 24-25 show the consistency of retention time and signal intensity of tagged peptides derived from HeLa digests at 33.3 minutes. This appeared to be consistent across the entire elution gradient.

[0040] [Figure 26] Figure 26 shows examples of increased precursor ion complexity for three peptides eluted at 19.45 minutes of the elution gradient.

[0041] [Figure 27] Figure 27 shows two examples of increased fragment ion complexity for two DIA selection windows with a width of 40 Da.

[0042] [Figure 28] Figure 28 shows the overall yeast protein abundance in a 6-plex DIA-tagged HeLA / yeast mixed proteome experiment, with quantitative values ​​derived from MS1 precursor intensity values. [Modes for carrying out the invention]

[0043] (Detailed explanation) (Set of mass labels)

[0044] The present disclosure provides a set of isotope-reactive tags having a mass difference in the range of millidaltons to several daltons for the purpose of labeling peptides and other biomolecules at a multiplexing rate that can greatly exceed 10-plex.

[0045] The present disclosure also provides a method of using isotope-reactive tags that enable a new form of analysis of labeled peptides, proteins, and other biomolecules, particularly for the discovery of biologically important differences between sets of biological samples.

[0046] In a first aspect, the present disclosure provides a set of two or more mass labels, each mass label having the formula:

Chemical formula

Chemical formula

[0047] The term "precise mass" means the theoretical mass of a mass label or coding site, and is the precise mass of the individual isotopes of the entire mass label or coding site, for example, 12 C = 12.000000, 13 C = 13.003355, 1 H = 1.007825, 16It is the sum of O = 15.994915. "Precise mass" takes mass defect into account.

[0048] In the literature, the term "isotopic" often refers to species that have different masses and are not coselectable by MS / MS; however, in the context of this disclosure, the term "isotopic" refers to species that have different masses but can still be coselected by MS / MS using DIA.

[0049] The exact mass difference between at least two of the mass labels in the set may be less than 100 millidaltons ("mDa"), less than 50 mDa, or less than 20 mDa. In some embodiments, the exact mass difference between at least two of the mass labels in the set is 2.5 mDa, 2.9 mDa, 6.3 mDa, 8.3 mDa, 9.3 mDa, or 10.2 mDa due to common isotopic substitution. For example, if the first label is 13 It contains the 1C isotope, and in the second label, this 13 C isotopes, 12 It has been replaced by C, 14 N isotope, 15 When replaced with the N isotope, the difference in precise mass between the two labels is 6.3 mDa.

[0050] In this specification, the term "label" is synonymous with the term "tag."

[0051] Those skilled in the art will understand that, in order to achieve the desired mass for each tag in the set, one or both of the sites X and M, the reactive functional group Re, or the analyte may be modified with heavy isotopes. Heavy isotopes are 2 H, 13 C, 15 N, or 18 You may choose from O.

[0052] In some embodiments, the total molecular weight of the mass label is 600 Da or less, for example, 500 Da or less, 400 Da or less, or 300 to 500 Da.

[0053] The mass labeling described herein is designed to react with biomolecules such as proteins to produce labeled biomolecules, such as labeled proteins.

[0054] In some embodiments, the optionally cleavable bond L includes, but is not limited to, an amide bond, a urea bond, an ester bond, or an ether bond. In some embodiments, the optionally cleavable bond L includes an amide bond. In some embodiments, the optionally cleavable bond L includes a urea bond. In some embodiments, the optionally cleavable bond L includes an ester bond. In other embodiments, the optionally cleavable bond L includes an ether bond. In some embodiments, the optionally cleavable bond L includes a carbon-carbon bond, as exemplified, for example, by the mass labeling in Example 1. In further embodiments, the optionally cleavable bond L is not cleavable by common fragmentation methods used in tandem mass spectrometry.

[0055] As used herein, the term “mass modifier M” means a region that ensures each mass label in the set has a desired mass. The mass modifier M is not necessarily detected by mass spectrometry. However, the mass modifier M may be detected as part of a complementary ion (see below). The mass modifier M is not particularly limited structurally and serves only to alter the total mass of the mass label.

[0056] In some embodiments, the mass modification section M has the following structure: [ka] (In the formula: Each R1 is independently H, a substituted or unsubstituted linear or branched C1-C6 alkyl group, a substituted or unsubstituted aliphatic cyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heterocyclic group, or an amino acid side chain. a is an integer between 0 and 10. b is at least 1, and c is at least 1. It has.

[0057] In some embodiments, the mass modifier M is: [ka] (In the formula: Each R1 is independently H, a substituted or unsubstituted linear or branched C1-C6 alkyl group, a substituted or unsubstituted aliphatic cyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heterocyclic group, or an amino acid side chain. a is an integer between 0 and 10. b is at least 1, c is at least 1, and * indicates an isotope mass regulation site, and oxygen is 18 Is it O, or is it carbon? 13 Is it C, or nitrogen? 15 N, or hydrogen, 2 (This represents H, and may contain one or more * symbols.) Selected from.

[0058] In the mass labeling provided for this disclosure, Re may be either a reactive functional group for attaching the mass labeling to the analyte or the analyte itself.

[0059] In some embodiments, the mass tag further includes a reactive functional group that enables the mass label to be conjugated to the analyte. The reactive functional group for attaching the mass label to the analyte is not particularly limited and may include any suitable reactive group.

[0060] The reactive functional group may react with amino groups on biomolecules, such as the ε-amino group of a lysine residue and the α-amino group of a peptide or protein. In the simplest embodiment, this may be an N-hydroxysuccinimide ester. Other reactive functional groups, such as those that react with thiol groups in biomolecules, are envisioned herein. In particular, these reactive functional groups are designed to react with thiol groups of cysteine ​​residues. Examples of reactive groups of this disclosure that can react with cysteine ​​residues are maleimide, haloacetyl, and 2-dithiopyridine groups. The thiol group of cysteine ​​undergoes nucleophilic addition to the double bond of the maleimide group and nucleophilic substitution with the haloacetyl or 2-dithiopyridine group.

[0061] Reactive functional groups capable of reacting with carbonyl or hydroxyl groups in biomolecules are also envisioned herein. In particular, these reactive functional groups are designed to react with carbonyl or hydroxyl groups of steroid hormones. The reactive groups of this disclosure capable of reacting with carbonyl or hydroxyl groups in biomolecules include hydrazides or (-CONH-CH2) n -ONH2 (wherein n is 1 to 6 (for example, n is 3, i.e., aminooxypropylamide)). These groups react with carbonyl groups to produce hydrazones or O-alkyloximes, respectively. Examples of reactive functional groups are shown in WO2011 / 036059, which is incorporated herein by reference.

[0062] In some embodiments, the reactive functional group is an N-hydroxysuccinimide ester, a 2,3,5,6-tetrafluorophenyl ester, or a sulfodichlorophenyl ester.

[0063] If Re is an analyte, the analyte may include, for example, amino acids, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, carbohydrates, lipids, phospholipids, or combinations thereof.

[0064] In some embodiments, the mass labels according to this disclosure and sets of two or more mass labels including heavy isotope mass series modifying groups are illustrated in the following exemplary embodiment 1.

[0065] (Embodiment 1)

[0066] Mass labeling is structure: [ka] Accurate mass: 440.26 Molecular formula:C 21 H 36 N4O6 (In the formula, * represents oxygen, 18 It is O, and carbon, 13 It is C, and nitrogen is 15 N is N, and hydrogen is 2 (This represents H, and may contain one or more * symbols.) It has.

[0067] In some embodiments, the mass modification site* is 13 C or 15 N is the set, and the set has the following structure: [ka] Label:6plexDIA-Δ0_440 Accurate mass: 440.26 Molecular formula:C 21 H 36 N4O6 [ka] Label: 6plexDIA-Δ4_444 Exact mass: 444.28 Molecular formula:[ 13 C]4C7H 36 N4O6 [ka] Label:6plexDIA-Δ8_448 Exact mass: 448.29 Molecular formula:[ 13 C]8C 13 H 36 N4O6 [ka] Label: 6plexDIA-Δ12_452 Exact mass: 452.28 Molecular formula:[ 13 C]9C 12 H 36 [ 15 N]3NO6 [ka] Label: 6plexDIA-Δ16_456 Accurate mass: 456.3 Molecular formula:[ 13 C] 13 C8H 36 [ 15 N]3NO6 [ka] Label: 6plexDIA-Δ20_460 Accurate mass: 460.31 Molecular formula:[ 13 C] 17 C4H 36 [ 15 N]3NO6 Includes a mass label with n=6.

[0068] (Embodiment 2)

[0069] The mass label has the following structure: [ka] It includes one of the following.

[0070] In some embodiments, the mass label may include any of the three structures described above in which one or more oxygen, carbon, nitrogen, and / or hydrogen components are replaced by isotopic substitution (for example, as illustrated above with respect to Embodiment 1). These options offer various levels of multiplexing and a range of sizes.

[0071] (Method of mass spectrometry)

[0072] Furthermore, the Disclosure also provides a method of mass spectrometry in which an analyte is quantified by mass spectrometry by identifying an ion having a mass-to-charge ratio equal to the mass of the analyte modified by the addition of one of the members of the set of isotopic mass labels, wherein the mass label is a mass label derived from the set of mass labels according to the Disclosure, and the identification of the analyte is obtained from data-independent acquired mass spectrometry.

[0073] In some embodiments, a mass spectrometry method includes: a) preparing a plurality of samples, each differentially labeled with an isotopic mass label or combination of isotopic mass labels, each selected from a set of two or more isotopic mass labels described herein; b) mixing the plurality of labeled samples to produce an analytical mixture containing labeled analytes; c) detecting the labeled analytes in a mass spectrometer; d) isolating one or more labeled analytes in the mass spectrometer based on one or more isolation windows of ≥1 Da, and then dissociating them to produce labeled analyte fragments; e) detecting the analyte fragments; and f) identifying the one or more labeled analytes based on the analyte fragments.

[0074] In some embodiments, the one or more isolation windows include isolation windows where ≤10Da, ≤20Da, ≤50Da, and / or ≤100Da.

[0075] In some embodiments, the labeled analyte may be identified based on the MS2 spectrum of the labeled analyte fragment and quantified using the MS1 spectrum of the labeled analyte.

[0076] In some embodiments, the dissociation in step d) is collision-induced dissociation in a mass spectrometer. In some embodiments, the dissociation is collision-induced dissociation in a mass spectrometer having a separation capability greater than 60,000 at a mass-to-charge ratio of 400, optionally, greater than 100,000 at a mass-to-charge ratio of 400, or greater than 250,000 at a mass-to-charge ratio of 400.

[0077] The analyte may be identified based on: i) the mass spectrum of the labeled analyte; or ii) the mass spectrum of an analyte fragment containing the mass label and / or intact mass label. If identification is performed by ii), the analyte fragment optionally contains a b-series ion (e.g., a b1 ion) containing the intact mass label.

[0078] Therefore, in some embodiments, the analyte may be identified based on the mass spectrum of the labeled analyte.

[0079] In some embodiments, the analyte may be identified based on the mass spectrum of an analyte fragment containing the mass label and / or intact mass label. In some embodiments, the analyte fragment containing the intact mass label is a b-series ion (e.g., a b1 ion) containing the intact mass label.

[0080] While a high-resolution mass spectrometer is required for the purpose of separating all possible tags described herein, the properties of such instrument are not particularly important for the implementation of the method described herein. In addition, many of the tags described in this application can still be separated even with an instrument having a resolution of only 1 Dalton, as long as a subset of possible tags that can be separated by a mass difference of 1 Dalton is used.

[0081] The mass labeling and methods described herein are further illustrated by the following examples. Such examples are illustrative and do not in any way limit the scope of the invention claimed herein, either here or during the proceedings of this application. [Examples]

[0082] (Examples)

[0083] (Example 1: Synthesis of 3-[3-[3-(diisobutylamino)propanoylamino]-propanoylamino]-propanoic acid (2,5-dioxopyrrolidine-1-yl) (diisobutyl-βAla-βAla-βAla-OSu))

[0084] (Step 1: Synthesis of 3-tert-butoxycarbonylaminopropionic acid (Boc-βAla-OH) (see Figure 1))

[0085] A solution of 16.32 g of sodium hydroxide in 163 mL of water is placed in a 2 L two-necked flask. 15.0 g of 3-aminopropanoic acid (β-alanine) and 150 mL of dioxane are added. A turbid solution is formed. Then, 53.47 g of di-tert-butyl dicarbonate ((Boc)2O) is added. An exothermic reaction (55-57°C) is observed, the pH stabilizes at approximately 9-10, and a solid precipitate forms. Stirring is continued for 5 hours. The mixture is allowed to stand overnight, and the next day 250 mL of water is added to form the resulting solution. The reaction solution is shaken thoroughly twice with 250 mL of diisopropyl ether. The diisopropyl ether phase is discarded. The aqueous phase is adjusted to pH 3 with 2 M hydrochloric acid (approximately 150 mL), and the product is extracted three times with 200 mL of dichloromethane. The organic phase was dried over magnesium sulfate, filtered, and concentrated to obtain 29.78 g of product (0.155 mol; 95.1%).

[0086] (Step 2: Synthesis of benzyl 3-aminopropionate hydrotosylate (H-βAla-OBn*pTosOH) (see Figure 2))

[0087] In a 1 L round-bottom flask fitted with a Dean-Stark apparatus and condenser, 28.51 g of 3-aminopropanoic acid (β-alanine), 199.64 mL of benzyl alcohol, 65.25 g of p-toluenesulfonic acid monohydrate-toluenesulfonic acid, and 400 mL of toluene were added. The reaction mixture was heated under reflux for 5 hours to separate approximately 11 mL of water, and then approximately 200 mL of toluene was removed by distillation. The solution was allowed to cool, and 400 mL of diisopropyl ether was slowly added at 45°C to 50°C, at which point the product began to crystallize. The mixture was stirred for 1.5 hours, and the temperature of the solution was reduced to 20°C to 25°C, completing the crystallization of the product. The solid was filtered, washed with diisopropyl ether, and dried under vacuum to obtain 111.08 g of the product (0.316 mol; 98.8%) as p-toluenesulfonate.

[0088] (Step 3: Synthesis of 3-[3-(tert-butoxycarbonylamino)propanoylamino]benzyl propanoate (Boc-βAla-βAla-OBn) (see Figure 3))

[0089] A suspension is obtained by adding 18.21 g of (1), 34.01 mL of diisopropylethylamine (DIPEA), 35.14 g of (2), and 21.44 g of 1-hydroxybenzotriazole monohydrate (HOBt) to approximately 200 mL of tetrahydrofuran in a 1 L two-necked flask. Upon addition of 24.92 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), the initial suspension becomes a solution within 15-20 minutes, and the temperature rises to approximately 30°C. The mixture is then stirred at 25°C for 7 hours and allowed to stand at room temperature overnight. The reaction solution is then concentrated using a rotary evaporator, and the residue is dissolved in 400 mL of ethyl acetate. This is then washed twice with 500 mL of saturated sodium bicarbonate solution and once with 500 mL of semi-saturated sodium chloride solution. The ethyl acetate phase is dried over MgSO4, filtered through silica gel, and concentrated. A solid white residue remains as the product, yielding 32.54 g (92.86 mmol; 96.7%).

[0090] (Step 4: Synthesis of 3-(3-aminopropanoylamino)propanoate benzyl hydrochloride (H-βAla-βAla-OBn*HCl) (see Figure 4))

[0091] 32.54 g of Boc-bAla-bAla-OBn is dissolved in 120 mL of dichloromethane, and 60 mL of 4 M HCl in dioxane is added. Gas generation begins, and stirring is carried out at 25°C for approximately 2 hours. The reaction is monitored by thin-layer chromatography (dichloromethane:methanol 20:1).

[0092] After completion, the solution was concentrated using a rotary evaporator and dried to quantitatively obtain the product (26.61 g, 100%).

[0093] (Step 5: Synthesis of 3-[3-[3-(tert-butoxycarbonylamino)propanoylamino]propanoylamino]benzyl propanoate (Boc-βAla-βAla-βAla-OBn) (see Figure 5))

[0094] A suspension is obtained by adding 17.57 g of (1), 31.58 mL of diisopropylethylamine (DIPEA), 26.61 g of (2), and 19.91 g of 1-hydroxybenzotriazole monohydrate (HOBt) to approximately 200 mL of tetrahydrofuran in a 1 L round-bottom flask. Upon addition of 23.14 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), the initial suspension becomes a solution within 20-30 minutes, and the temperature rises to approximately 35°C. The mixture is then stirred at 25°C for 5 hours and allowed to stand overnight at room temperature. The reaction solution is then concentrated using a rotary evaporator, and the residue is dissolved in 400 mL of ethyl acetate. This is then thoroughly shaken twice with 400 mL of saturated sodium bicarbonate solution and once with 500 mL of semi-saturated sodium chloride solution. The ethyl acetate phase is dried with MgSO4 and concentrated. During concentration, the product crystallizes, yielding 36.31 g (86.15 mmol, 92.8%).

[0095] (Step 6: Synthesis of 3-[3-(3-aminopropanoylamino)propanoylamino]benzyl hydrochloride (H-βAla-βAla-βAla-OBn*HCl) (see Figure 6))

[0096] 3.37 g of Boc-βAla-βAla-βAla-OBn is dissolved in 10.4 mL of dichloromethane, and 5.2 mL of 4 M HCl in dioxane is added. Gas generation begins, and stirring is carried out at 25°C for approximately 2 hours. The reaction is monitored by thin-layer chromatography (dichloromethane:methanol 20:1). After completion, the solution is concentrated using a rotary evaporator and dried to quantitatively obtain the product (2.86 g, 100%).

[0097] (Step 7: Synthesis of 3-[3-[3-(diisobutylamino)propanoylamino]propanoylamino]benzyl propanoate (diisobutyl-βAla-βAla-βAla-OBn) (see Figure 7))

[0098] 2.86 g of H-βAla-βAla-βAla-OBn*HCl is stirred with 30 mL of 1,2-dichloroethane under argon. Then, 5.44 mL of diisopropylethylamine and 1.83 mL of isobutyraldehyde are added, and the mixture is stirred for 30 minutes to obtain a clear solution. Subsequently, 6.78 g of sodium triacetoxyborohydride is added, and the temperature rises to approximately 40°C. Stirring is continued overnight at 25°C. Then, approximately half of the 1,2-dichloroethane is removed using a rotary evaporator, and 50 mL of ethyl acetate is added to the residue. The organic layer was washed twice with 100 mL of saturated sodium bicarbonate solution and once with 100 mL of semi-saturated sodium chloride solution, dried over MgSO4, filtered through silica gel using approximately 500 mL of ethyl acetate and approximately 500 mL of ethyl acetate:methanol 10:1, and concentrated to obtain 3.35 g of product as a solid (7.72 mmol, 96.6%).

[0099] (Step 8: Synthesis of 3-{3-[3-(diisobutylamino)propionylamino]propionylamino}propanoic acid (diisobutyl-βAla-βAla-βAla-OH) (see Figure 8))

[0100] 0.17 g of 5%-Pd / C catalyst and 20 mL of methanol are placed in a hydrogenation apparatus under argon. Then, 3.35 g of diisobutyl-βAla-βAla-βAla-OBn dissolved in 20 mL of methanol is added, and hydrogenation is carried out at 25°C until hydrogen uptake is complete. The reaction mixture is filtered through a decalite filter aid to remove the Pd / C catalyst, and the filtrate is washed with methanol. The filtrate is concentrated under vacuum until dry. The oily residue is crystallized using ethyl acetate:diisopropyl ether 2:3 to obtain 1.78 g of product as a white powder (5.19 mmol, 67%).

[0101] (Step 9: Synthesis of 3-[3-[3-(diisobutylamino)propanoylamino]propanoylamino]propanoic acid (2,5-dioxopyrrolidine-1-yl) (diisobutyl-βAla-βAla-βAla-OSu) (see Figure 9))

[0102] In a 100 mL flask, dissolve 1.29 g of diisobutyl-βAla-βAla-βAla-OH in 25 mL of dichloromethane at room temperature. Then, add 1.25 g of N,N′-disuccinimidyl carbonate (DSC). The initial suspension will dissolve while releasing CO2. After the release of CO2 is complete, stir the reaction mixture for 1 hour. Then, dilute the reaction solution to 50 mL with dichloromethane and shake thoroughly three times with 40 mL of 4:1 saturated sodium bicarbonate / saturated sodium chloride solution, and once with 40 mL of semi-saturated sodium chloride solution. Dry the dichloromethane phase over MgSO4, filter, and concentrate using a rotary evaporator.

[0103] The residue is dissolved in 5 mL of ethyl acetate at approximately 65°C. The product crystallizes during cooling. The crystals are aspirated on a reverse frit under an argon atmosphere, washed with ethyl acetate and diisopropyl ether, and dried at room temperature under vacuum to obtain 0.81 g of the product as a white powder (1.84 mmol, 49%).

[0104] It will be apparent to those skilled in the art that by using heavily isotopically labeled versions of β-alanine and isobutyraldehyde, these can be substituted in the synthesis steps described above to produce different versions with multiple different isotopic masses. Figures 10-17 show the results of mass spectra for six sets of mass labels according to this embodiment (i.e., those shown above by chemical structure in Embodiment 1) with different isotopic substitutions.

[0105] (Example 2: Synthesis of five additional isotopic variants of 3-[3-[3-(diisobutylamino)propanoylamino]propanoylamino]propanoic acid (2,5-dioxopyrrolidine-1-yl) (diisobutyl-βAla-βAla-βAla-OSu) (6plexDIA-Δ0_440))

[0106] Following the synthesis of undoped 6plexDIA-Δ0_440, the inventors repeated the synthesis using different precursors doped at different C and N positions, as shown in Embodiment 1. The synthesis conditions were identical in all other respects. The final product was introduced into a TSQ Vantage mass spectrometer [Thermo Scientific] to evaluate the precursor mass and peak purity (Figures 10-17). All tags showed the expected mass and very good purity.

[0107] (Example 3: Analysis of BSA digests using 6plexDIA-Δ0_440)

[0108] To demonstrate protein labeling, the inventors used 150 pmol of bovine serum albumin trypsin digest [New England Biolabs P8108S]. The peptide was labeled with a 6plexDIA-Δ0_440 tag by incubation in 100 mM TEAB buffer for 1 hour. The labeling was quenched by adding a 0.25% hydroxylamine solution, and the labeled peptide was purified using StageTip. The eluate was completely dried using a SpeedVac centrifuge. In parallel, the inventors prepared unlabeled samples and Tandem Mass Tag® (TMT®) [Proteome Sciences plc]-labeled samples for comparison.

[0109] All three BSA digests were analyzed using an Orbitrap Fusion® Tribrid® mass spectrometer [all Thermo Scientific] with an online EASY-nLC® 1200 UHPLC chromatography system, and spectra were collected in data-dependent acquisition mode. As expected, the retention time of the 6plexDIA-Δ0_440 peptide was slower than that of the TMT® or unlabeled equivalent peptide (Figure 18). The MS / MS spectra of representative unlabeled BSA peptides (Figure 19) were compared with those of the 6plexDIA-Δ0_440 labeled equivalent (Figure 20), and sufficient structural ions were observed to enable reliable sequence assignment.

[0110] (Example 4: Analysis of HeLa digests using the 6-plex DIA tag of Embodiment 1)

[0111] Six digests of a human HeLa cell line [Pierce Biotechnology] were independently labeled with one of the 6plex DIA tags of Embodiment 1, essentially as described in Example 3. After quenching the labeling reaction, the six digests were pooled to obtain a single HeLa analytical sample. The HeLa analytical sample was analyzed by LC-MS / MS on an Orbitrap Exploris® 480 [all Thermo Scientific] using an online EASY-nLC® 1200 UHPLC system and a FAIMS Pro source. Peptides were eluted over 45 minutes using an active acetonitrile gradient. MS1 survey scans were collected at a resolution of 120,000, and MS / MS spectra were acquired in DDA mode.

[0112] Data analysis revealed that, compared to the unlabeled digest, the 6plex DIA tag had a higher proportion of +3 ions, while simultaneously showing a decrease in +2 ions, although they still accounted for the largest proportion. Similar increases were observed for +4 and +5 ions, although their abundances remained relatively low. It is also noteworthy that the median precursor ion intensity of +3 ions was higher in the labeled digest compared to the unlabeled +3 ion. Examples for unlabeled and labeled HeLa digests are shown in Figure 21.

[0113] Furthermore, the +3 ion exhibited a higher identification success rate than all other charge states, with a maximum of approximately 43% achieved at a normalized collision energy of 35% (Figure 22). All six tags showed very similar labeling efficiencies in the HeLa digest, with the percentage of labeled peptides ranging from 98.9% to 99.6% (Figure 23). The inventors also confirmed that all six different tagged peptide forms co-eluted completely (an example for one peptide is shown in Figure 24) and that the median precursor ion intensities of all six labeled digests were very similar (Figure 25).

[0114] (Example 5: Analysis of BSA digest using the 6-plex DIA tag of Embodiment 1)

[0115] Six digests of BSA were independently labeled with one of the 6plex DIA tags of Embodiment 1, essentially as described in Example 3. After quenching the labeling reaction, the six digests were pooled to obtain a single BSA analytical sample, which was analyzed by LC-MS / MS using an Orbitrap Exploris® 480 mass spectrometer (Thermo Scientific) with an online EASY-nLC® 1200 UHPLC system. Peptides were eluted over 45 minutes using an active acetonitrile gradient. MS1 survey scans were collected at a resolution of 60,000, and MS / MS spectra were acquired in DIA mode with an isolation window of 10 or 40 Da.

[0116] Analysis of the data revealed an expected increase in precursor ion intensity, as shown for the survey scan at 19.4 min, and the overlap of the isotopic envelope depended on the charge state and the number of attached DIA tags (Figure 26). An expected increase in complexity was also observed in the fragmentation spectra of the 40 Da isolation window MS / MS scan (Figure 27). This was particularly evident in lysine-terminated peptides, where both the β- and γ-series ions showed higher complexity. For peptides terminated with any other amino acid, the γ- ion did not show increased complexity, while the corresponding β- ion exhibited six variants attributable to the presence of DIA tags.

[0117] (Example 6 - Analysis of HeLa digest spiked with yeast digest and labeled with the 6-plex DIA tag of Embodiment 1)

[0118] Six distinct samples were prepared by spike-spiking 195 μg of HeLa digest with six different amounts of yeast digest (0%, 2%, 3.5%, 5.8%, 9.7%, and 15.6%). Each sample was individually mixed with six 6plexDIA reagents (Table 1) at a final concentration of 16.5 mM and incubated at room temperature for 1 hour with shaking. The reaction was stopped by adding 0.25% hydroxylamine at room temperature for 15 minutes. 25% of each labeled peptide sample was purified by tandem solid-phase extraction (Oasis HLB (Waters) and SCX (SP Sepharose Fast Flow, Sigma)), and the eluate was completely dried using a SpeedVac centrifuge. 75% of each labeled peptide was pooled to prepare one 6plex, and then 50% of this mixed 6plex was purified by tandem SPE (HLB / SCX) as described above and dried under vacuum. [Table 1] (Table 1. Assignment of 6plex DIA tags to HeLa+ yeast digest samples)

[0119] Peptides were analyzed by LC-MS / MS using an Orbitrap Exploris® 480 mass spectrometer (Thermo Scientific) with an online EASY-nLC® 1200 UHPLC system. Peptides were eluted with an active acetonitrile gradient for 110 minutes. MS1 survey scans were collected at a resolution of 60K, and MS / MS spectra were acquired in DIA mode with a 10 Da isolation window. MS raw data files were searched using DIA-NN software with an in silico-generated reference library. The inventors identified 3,584 human and yeast proteins throughout the sixplex samples and confirmed the expected increase in the abundance of yeast peptide precursors and fragments consistent with spike concentrations in samples containing 5.8% or more yeast (Figure 28).

Claims

1. A set of two or more isotopic mass labels, each label having the formula: 【Chemistry 1】 (In the formula: X is a coding region having precise mass, L is a bond that can be arbitrarily broken by collision in a mass spectrometer. M is a mass modifier, Re is a reactive functional group for attaching the mass label to the analyte, or the analyte itself, and X is given by the following general formula: 【Chemistry 2】 (including) A set of two or more isotopic mass labels, including the set of two or more isotopic mass labels.

2. A set of two or more isotopic mass labels, Each mass label is given by the formula: 【Transformation 3】 (In the formula: X is a coding region having precise mass, L is a bond that can be arbitrarily broken by collision in a mass spectrometer. M is a mass modifier, and A is the analyte. including and Each mass marker in the set has a different integer mass, and X is given by the following general formula: 【Chemistry 4】 A set of two or more isotopic mass labels, including the set of two or more isotopic mass labels.

3. The mass modification section M has the following structure: 【Transformation 5】 (In the formula: Each R 1 These are independently H, substituted or unsubstituted linear or branched C 1 -C 6 Alkyl groups, substituted or unsubstituted aliphatic cyclic groups, substituted or unsubstituted aromatic groups, or substituted or unsubstituted heterocyclic groups, or amino acid side chains. a is an integer between 0 and 10. b is at least 1, and c is at least 1. A set of two or more isotopic mass labels according to claim 1 or 2, comprising:

4. The mass modification section M has the following structure: 【Transformation 6】 (In the formula: Each R 1 These are independently H, substituted or unsubstituted linear or branched C 1 -C 6 Alkyl groups, substituted or unsubstituted aliphatic cyclic groups, substituted or unsubstituted aromatic groups, or substituted or unsubstituted heterocyclic groups, or amino acid side chains. a is an integer between 0 and 10. b is at least 1, c is at least 1, and * is an isotope mass adjustment site, and oxygen is 18 O, or carbon is 13 C, or nitrogen is 15 N, or hydrogen is 2 H, and (One or more * symbols may be present.) A set of two or more isotopic mass labels according to claim 1 or 2, comprising:

5. Each mass label has the following structure: 【Transformation 7】 (In the formula, * represents oxygen, 18 It is O, and carbon, 13 It is C, and nitrogen is 15 (This represents N, and may contain one or more * symbols.) A set of two or more isotopic mass labels according to any one of claims 1 to 4, comprising:

6. The aforementioned set has the following structure: 【Transformation 8】 A set of two or more isotopic mass labels according to any one of claims 1 to 5, comprising n=6 mass labels having .

7. A set of two or more isotopic mass labels according to any one of claims 1 to 5, wherein the difference in precise mass between at least two of the mass labels is less than 100 millidaltons ("mDa"), less than 50 mDa, or less than 20 mDa.

8. A set of two or more isotopic mass labels according to any one of claims 1 to 5, wherein the difference in precise mass between at least two of the mass labels is 2.5 mDa, 2.9 mDa, 6.3 mDa, 8.3 mDa, 9.3 mDa, or 10.2 mDa.

9. a) Preparing a plurality of samples, wherein each sample is differentially labeled with an isotopic mass label or combination of isotopic mass labels, each selected from a set of two or more isotopic mass labels described in any one of claims 1 to 8; b) Mixing the multiple labeled samples to produce an analytical mixture containing the labeled analyte; c) Detecting the labeled analyte using a mass spectrometer; d) In the mass spectrometer, one or more labeled analytes are isolated based on one or more isolation windows of ≥1 Dalton ("Da"), and then dissociated to produce labeled analyte fragments; e) detecting the analyte fragment; and f) Identifying one or more labeled analytes based on the analyte fragment, A method of mass spectrometry, including [specific data / method].

10. The method according to claim 9, wherein the one or more isolation windows include an isolation window where ≤ 10Da.

11. The method according to claim 9, wherein the one or more isolation windows include an isolation window where ≤20Da.

12. The method according to claim 9, wherein the one or more isolation windows include an isolation window where ≤ 50 Da.

13. The method according to claim 9, wherein one or more isolation windows include an isolation window where ≤ 100 Da.

14. The method according to any one of claims 9 to 13, wherein the dissociation in step d) is collision-induced dissociation in a mass spectrometer.

15. The method according to any one of claims 9 to 14, wherein the dissociation is collision-induced dissociation in a mass spectrometer having a separation capability of more than 60,000 at a mass-to-charge ratio of 400, optionally, more than 100,000 at a mass-to-charge ratio of 400, or more than 250,000 at a mass-to-charge ratio of 400.

16. The method according to any one of claims 9 to 15, wherein the labeled analyte may be identified based on the MS2 spectrum of the labeled analyte fragment and quantified using the MS1 spectrum of the labeled analyte.

17. The method according to any one of claims 9 to 16, wherein each isotopic mass label is separated from all other tags in the set by a difference of at least 0.003 Da, 0.006 Da, or at least 1 Da.

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