Methods for non-targeted identification of peptide sequences

Abstract

The disclosure describes a method for non-targeted identification of a peptide sequence in a peptide comprising 2 to 6 amino acids present in a sample, the method being implemented by a device comprising processing circuitry and a memory, the memory comprising: at least one predetermined library of peptides comprising 2 to 6 amino acids, each predetermined peptide being defined by a peptide sequence; and a set of information respectively associated with a peptide present in the sample, the peptides in the sample being referred to as measured peptides, the measured peptides and the predetermined peptides being labeled at the same end with the same end label. A method for analyzing peptides comprising 2 to 6 amino acids in a sample is also described, the method comprising a data processing step implementing a method for non-targeted identification of a peptide sequence.

Description

Technical Field

[0001] This invention relates to a method for non-targeted identification of peptide sequences comprising 2 to 6 amino acids present in a sample, the method being implemented by a device comprising a processing circuit system and a memory, and further to a method for analyzing peptides in a sample. The invention also relates to a method for analyzing peptides having 2 to 6 amino acids in a sample using the identification method of the invention. Background Technology

[0002] Due to their physicochemical properties and potential activity, small peptides derived from proteins have attracted significant attention from the food, pharmaceutical, and cosmetic industries. However, the enzymatic hydrolysis of proteins produces a complex mixture of peptides with various sizes and properties.

[0003] The analysis of total peptide / protein content and free amino acid content in samples is well-established. Protein analysis using proteomics is also known.

[0004] However, conventional proteomics or peptide analysis methods do not cover small peptides, and due to their small size and wide variation in physicochemical properties, they are difficult to separate by liquid chromatography (LC) and detect by mass spectrometry (MS). Therefore, more detailed characterization of small peptides in complex mixtures remains a challenge.

[0005] The applicant typically develops yeast extracts: food matrices obtained through the hydrolysis of yeast extract. During the hydrolysis step, proteins in the yeast extract are broken down into a complex mixture containing free amino acids and peptides with 2 to 35 amino acids. Yeast extract proteins can be analyzed using proteomics analysis methods. Peptidomics analysis techniques also exist for characterizing peptides containing at least 5 or 6 amino acids. However, current techniques do not allow for the rapid characterization of all short or small peptides in yeast extract samples, even though these small peptides constitute the major components of most yeast extracts.

[0006] Therefore, there is a need for a method that can systematically identify all small peptides in a sample without targeting. Summary of the Invention

[0007] A method for non-targeted identification of peptide sequences comprising 2 to 6 amino acids present in a sample is proposed, the method being implemented by a device comprising a processing circuit system and a memory, the memory comprising: - At least one predetermined peptide library, wherein the predetermined peptides comprise 2 to 6 amino acids, each predetermined peptide being defined by a peptide sequence, and for each predetermined peptide, the library comprising: The constituent amino acids of the peptide sequence and the total mass-to-charge ratio of the peptide sequence (m / z The peptide sequence has at least one terminal amino acid, and the at least one terminal amino acid is labeled with a terminal tag. Fragment ions calculated based on the peptide sequence, each associated with a mass-to-charge ratio, and at least one or more of the calculated fragment ions containing the terminal label. - Information sets, each associated with a peptide (referred to as an analyte) present in the sample as measured by mass spectrometry, each analyte comprising a peptide sequence having at least one terminal amino acid with a terminal label, each information set of the analyte comprising at least one MS / MS spectrum, the at least one MS / MS spectrum comprising the total mass-to-charge ratio of the analyte and one or more mass-to-charge ratios of the analyte fragment ions. The test peptide and the predetermined peptide are labeled with the same end marker at the same end. For each information set of the peptide being tested, the method includes: / a / Based on the library and based on the at least one MS / MS spectrum, the selection of predetermined peptides is determined, wherein the total mass-to-charge ratio of each selected predetermined peptide is equal to the total mass-to-charge ratio of the analyte peptide, and the error tolerance is lower than a predetermined value. / b / The labeled terminal amino acid of the analyte peptide is determined by comparing the mass-to-charge ratio of the analyte fragment ion with the calculated mass-to-charge ratio of the fragment ion of the selected predetermined peptide, provided that the error tolerance is below a predetermined value. / c / Based on the calculated fragment ions in the selection, the determined labeled terminal amino acids, and the fragment ions to be tested, at least one reconstructed peptide sequence of the peptide to be tested is determined. - Deliver an identification list that identifies the analyte peptide in the sample, wherein each analyte peptide in the list is defined by a reconstructed peptide sequence.

[0008] Advantageously, this allows for the acquisition of an identification list as an output of the method without prior assumptions about the structure and composition of the various peptides contained in the sample. The list comprises all peptides in the sample having a peptide sequence of 2 to 6 amino acids in length (or number of amino acids), typically 2 to 4 amino acids. As will be detailed below, the peptides present in the sample may contain modified amino acids, particularly modified amino acids containing modified side chains, wherein the modifications may be of natural or synthetic origin.

[0009] Each fragment ion being tested can indicate the presence of one or more linked amino acids within the peptide sequence of the peptide being tested.

[0010] The total mass-to-charge ratio can be understood as referring to the mass-to-charge ratio of the entire peptide being tested, which means that the total mass-to-charge ratio includes all constitutive amino acids with terminal tags in the peptide sequence of the peptide being tested.

[0011] In one or more embodiments, the library may be organized into two or more different libraries. More specifically, the library data may be distributed across one or more different libraries.

[0012] According to one or more examples, the comparison in / b / may involve comparing the mass-to-charge ratio of the analyte fragment ion with the terminal label (detected in the MS / MS spectrum of the analyte peptide) with the mass-to-charge ratio of the selected fragment ions and / or the mass-to-charge ratio of the selected fragment ions with the terminal label. This comparison may be performed with an error tolerance below a predetermined value.

[0013] In one or more embodiments, the calculated fragment ions can be calculated based on the peptide sequence by applying a fragmentation method to the peptide sequence.

[0014] According to one or more embodiments, the fragmentation method may be defined by one or more fragmentation rules, which may be included in a fragmentation rule library. The calculated fragment ions can be calculated based on a predetermined peptide by applying fragmentation rules from the library.

[0015] In one or more embodiments, the fragmentation method may be the same as the fragmentation method of the mass spectrometer.

[0016] In one or more embodiments, the calculated fragment ions (or a set of calculated fragment ions) can be calculated based on a predetermined peptide by applying multiple fragmentation methods (e.g., fragmentation methods defined in the fragmentation rule library). More specifically, multiple sets of calculated fragment ions can be calculated based on predetermined peptides for which multiple fragmentation methods are applied respectively, wherein each set of calculated fragment ions is obtained based on a corresponding fragmentation method.

[0017] Therefore, advantageously, the same library can be used to identify peptide sequences in a sample that have been analyzed by different mass spectrometers or by the same mass spectrometer containing multiple mass analyzers, preferably by mass spectrometers capable of tandem analysis using specific fragmentation methods or capable of being measured by different fragmentation methods.

[0018] In one or more embodiments, the predetermined peptide library contains all possible peptide sequences consisting of 2 to 6 amino acids obtained from a set of N amino acids; each sequence in the predetermined library defines a predetermined peptide of the library, and further contains a terminal amino acid with the terminal label.

[0019] The term "all possible peptide sequences" can be understood to mean all possible combinations that form a peptide sequence of 2 to 6 amino acids in length, with the terminal amino acid bearing the terminal label, which is obtained from a set of N amino acids.

[0020] In one or more embodiments, N may comprise at least 22 protein-derived amino acids and one or more modified amino acids. In one or more embodiments, the modified amino acids may be modified starting from protein-derived amino acids. In one or more embodiments, N may be greater than 2, preferably greater than 20. In one or more embodiments, N may be less than 500, preferably less than 400, more preferably less than 300, and even more preferably less than 200. N can typically be between 2 and 100, for example, between 2 and 50.

[0021] In one or more embodiments, the library contains at least one predetermined peptide, which represents a plurality of charged predetermined peptides, each charged predetermined peptide having the same peptide sequence as the at least one predetermined peptide and a corresponding charge state.

[0022] A pre-charged peptide is understood to mean a pre-charged peptide having one or more charge states selected from: one or more positive charges (e.g., +1, +2, or +3) or one or more negative charges (e.g., -1, -2, -3).

[0023] Therefore, advantageously, the peptide sequence of the test peptide carrying one or more charges (e.g., single-charged, double-charged, or triple-charged) can be determined.

[0024] In one or more embodiments, the ends with the terminal marker are identical for all peptide sequences in a predetermined library.

[0025] In one or more embodiments, the type of the labeled terminal amino acid terminus is: N End and / or C The end, preferably N End.

[0026] In one or more embodiments, the total mass-to-charge ratio of the predetermined peptide ( m / z The mass-to-charge ratio is determined based on the constitutive amino acid residues in the sequence of the predetermined peptide.

[0027] In one or more embodiments, the mass spectrometer is a mass spectrometer (MS / MS) with tandem analysis capability, preferably a mass spectrometer coupled with liquid chromatography (LC).

[0028] In one or more embodiments, in / c / , at least one peptide sequence of the peptide being tested is reconstructed by the following: - The identified terminal amino acid of the label, - A comparison between the mass-to-charge ratio of the fragment ion being measured and the calculated mass-to-charge ratio of the fragment ion.

[0029] The comparison between the mass-to-charge ratio of the measured fragment ion and the calculated mass-to-charge ratio of the fragment ion can be performed while taking into account an error tolerance lower than a predetermined value.

[0030] In one or more embodiments, the memory includes at least a first library and a second library, the first library containing the constitutive amino acids of the peptide sequence and the total mass-to-charge ratio of the peptide sequence. m / z The peptide sequence has at least one terminal amino acid, the at least one terminal amino acid being labeled with the terminal tag; and the second library is determined only for the selected predetermined peptides, the second library containing at least the selected predetermined peptides and containing fragment ions, the fragment ions being calculated based on the peptide sequence of the selected predetermined peptides, the calculated fragment ions being associated with mass-to-charge ratios, and at least one or more of the calculated fragment ions containing the terminal tag.

[0031] Therefore, the computation time required to determine the reconstructed peptide sequence can be advantageously reduced.

[0032] In one or more embodiments, the second library may be determined based on at least one fragmentation method applied to the predetermined peptide in the selection.

[0033] In one or more embodiments, each information set associated with the test peptide may also include mass spectrometry information (referred to as MS spectrum), which may include the mass-to-charge ratio, retention time, and intensity of the test peptide (i.e., intensity of the precursor ion) of the test peptide (i.e., precursor ion).

[0034] The retention times included in the MS spectrum (or information set) of the peptide being tested can be obtained based on measurements performed after separation by liquid chromatography.

[0035] In one or more embodiments, the selection may alternatively be determined based on information in the MS spectrum of the peptide being tested and information in the library.

[0036] In one or more embodiments, the peptide sequence or multiple peptide sequences reconstructed for each test peptide can be derived from what is known as "de novo" (DNOW). de novo The sequencing method used in sequencing is determined by the sequencing method itself.

[0037] The peptide sequence of the peptide to be tested is reconstructed by applying the methodological principles used in "de novo" sequencing, utilizing the MS / MS spectrum of the peptide to be tested, the identified labeled terminal amino acids, and the calculated fragment ions of the selected predetermined peptide.

[0038] In one or more embodiments, when multiple reconstructed peptide sequences are determined for the same test peptide in / c / , the method further comprises: / d / A ​​score is calculated for each reconstructed peptide sequence based on the information set associated with the peptide being tested and based on the reconstructed peptide sequence. The reconstructed peptide sequence of the test peptide was selected based on a comparison of the scores of the reconstructed peptide sequences, and the peptide sequence with the highest score was selected.

[0039] Therefore, in the case of peptide co-elution, using a scoring method is advantageous in ensuring that the peptide sequence determined for the test peptide does indeed correspond to the test peptide and to the information set obtained for the test peptide.

[0040] In one or more embodiments, each mass-to-charge ratio of the analyte fragment ion is correlated with its intensity in the MS / MS spectrum, and the score for each reconstructed peptide sequence of the same analyte peptide is determined based on the following equation:

[0041] In one or more embodiments, the peptide sequence score is calculated using only the tested fragment ions with the terminal label.

[0042] In one or more embodiments, in / b / , one or more labeled terminal amino acids are identified, and in / c / , for each identified labeled terminal amino acid, at least one reconstructed peptide sequence of the test peptide is determined based on the calculated fragment ion in the selection, the identified labeled terminal amino acid, and the test fragment ion.

[0043] In one or more embodiments, the predetermined value of the error tolerance is less than 20 ppm, preferably less than 15 ppm, more preferably less than 10 ppm, and even more preferably less than 5 ppm.

[0044] In one or more embodiments, the list may include retention times associated with the test peptide, each retention time being obtained from the information set of the reconstructed peptide sequence used to determine the test peptide, particularly, for example, the MS spectrum.

[0045] Each peptide in the list can be associated with a corresponding retention time obtained from the information set (e.g., MS spectrum) of the reconstructed peptide sequence used to determine the peptide.

[0046] In one or more embodiments, each test peptide in the list is associated with the information set of the reconstructed peptide sequence used to determine the test peptide by means of identification.

[0047] Advantageously, the use of identification methods allows for rapid sorting of the information set and ensures that the information set (e.g., one or more MS / MS spectra and / or MS spectra) is properly correlated with the reconstructed peptide sequence used. Furthermore, the identification methods facilitate post-processing of the obtained data (e.g., identification lists, information sets, etc.).

[0048] In one or more embodiments, the list contains intensities associated with each of the test peptides, each intensity obtained from an information set (e.g., MS spectroscopy) of the reconstructed peptide sequence used to determine the test peptide, and each test peptide in the list contains a proportion value indicating the proportion of the peptide in the mixture. The proportion value "Qp" for each test peptide in the list is calculated based on the following:

[0049] Therefore, it is advantageous to obtain the relative proportions of the peptides in the mixture.

[0050] The intensity of each peptide in the list can be obtained from the MS spectrum of the peptide included in the information set.

[0051] In one or more embodiments, the peptide is a natural peptide and / or a synthetic peptide.

[0052] In one or more embodiments, the peptide contains at least one labeled terminal amino acid and one or more amino acids selected from protein-derived amino acids or modified amino acids.

[0053] In one or more embodiments, the labeled terminal amino acid is marked with N End-marked N terminal amino acids, the N The terminal label is selected from dansyl, dabsyl, 1-naphthyl isocyanate (IsoC), or 1-naphthyl isothiocyanate, each of which is optionally marked with carbon-13 ( 13 C) Deuterium (D) Nitrogen-15 ( 15 N) and / or sulfur-34 ( 34 One or more atomic markers in S). These markers allow for differentiation between the terminal markers.N The terminal label can be selected, for example, from dansyl, with one or more carbon-13 ( 13 C) Atom-labeled dansyl or dansyl, dabusulfonyl, 1-naphthyl isocyanate (IsoC) or 1-naphthyl isothiocyanate labeled with deuterium (D).

[0054] In one or more embodiments, the labeled terminal amino acid is marked with C End-marked C terminal amino acids, the C End markings are selected from 2-( N,N' -dimethylamino)ethyl-1-amine (DMED) or 3-( N,N' -dimethylamino)prop-1-amine (DMAPA), each of these terminal tags is optionally represented by carbon-13 ( 13 C), deuterium (D) and / or nitrogen-15 ( 15 One or more atomic markers in N). These markers allow for differentiation between the terminal markers. C The end marker can be selected, for example, from DMED or DMAPA.

[0055] In one or more embodiments, the peptide has undergone a protective treatment to block the free thiol group on an amino acid (such as cysteine), typically an alkylation treatment, wherein the predetermined mass-to-charge ratio of the peptide in the library takes into account the protective treatment to block the free thiol group of cysteine. This protective treatment is typically performed by carbamoyl methylation, usually using chloroacetamide or iodoacetamide. This protective treatment can also be performed using other reactants known to those skilled in the art, such as β-(4-hydroxyphenyl)ethyliodoacetamide, iodoacetic acid, and... N -Ethylmaleimide is used.

[0056] In one or more embodiments, the liquid chromatography method is reversed-phase liquid chromatography, preferably performed using a gradient of at least one hour.

[0057] In one or more embodiments, the mass spectrometer is equipped with a mass analyzer, preferably selected from quadrupole ion trap mass analyzers and triple quadrupole mass analyzers; more preferably, the mass spectrometer is equipped with a high-resolution mass analyzer, selected from orbital trap analyzers, Q-TOF analyzers, TIMS-TOF analyzers, and FT-ICR analyzers.

[0058] In one or more embodiments, the peptide present in the sample is a natural peptide and / or a synthetic peptide.

[0059] In one or more embodiments, the sample is a mixture of natural small peptides or a mixture of peptides obtained through peptide synthesis. The sample is preferably a protein hydrolysate, such as yeast protein hydrolysate, plant protein hydrolysate, or animal protein hydrolysate.

[0060] According to another aspect, a computer program is provided that includes instructions for implementing the method according to the present disclosure when the program is executed by a processor.

[0061] According to another aspect, a non-transitory computer-readable storage medium is provided, wherein a program is stored for implementing the method according to the present disclosure when the program is executed by a processor.

[0062] According to another aspect, a method for analyzing peptides having 2 to 6 amino acids in a sample is proposed, the method comprising: i) A sample processing step, the sample processing step comprising at least one step i1) of labeling the ends of the peptides in the sample by contacting the sample with an end label. ii) Analytical steps, wherein the analytical steps utilize liquid chromatography coupled with a mass spectrometer, preferably a mass spectrometer (MS / MS) with tandem analytical capabilities. iii) Data processing steps, which implement the methods for non-targeted identification of peptide sequences as described in this document.

[0063] In one or more embodiments, the sample processing step further includes alkylating the peptide by contacting the peptide in the sample with an alkylating agent, and the alkylation step is preferably performed before the sample processing step.

[0064] In one or more embodiments, the liquid chromatography method is reversed-phase liquid chromatography, preferably performed using a gradient of at least one hour.

[0065] In one or more embodiments, the mass spectrometer is equipped with a mass analyzer, which is preferably a quadrupole ion trap mass analyzer or a triple quadrupole mass analyzer, and more preferably, a high-resolution mass analyzer selected from orbital trap analyzers, Q-TOF analyzers, TIMS-TOF analyzers, and FT-ICR analyzers.

[0066] In one or more embodiments, the sample contains protein hydrolysates, such as yeast protein hydrolysates, plant protein hydrolysates, or animal protein hydrolysates. Attached Figure Description

[0067] Other features, details, and advantages will become apparent from reading the following detailed description and analyzing the accompanying drawings, in which: Figure 1 [ Figure 1 The flowchart illustrates a method for non-targeted identification of peptide sequences from a sample.

[0068] Figure 2 [ Figure 2 This illustration schematically shows an example of a pre-defined peptide library.

[0069] Figure 3 [ Figure 3 This schematically illustrates the co-elution phenomenon of two peptides.

[0070] Figure 4 [ Figure 4 The apparatus for implementing the methods of this disclosure is shown.

[0071] [ Figure 5 This figure shows the number of short peptide sequences identified using the method of the present invention in various protein hydrolysates derived from soybean (SH), casein (CH), and yeast (YE). Values ​​represent the mean of three replicates ± standard deviation.

[0072] [ Figure 6 The image shows the soybeans (SH, Figure 6 A) Casein (CH, Figure 6 B) and yeast (YE, Figure 6 Comparative qualitative analysis of short peptide sequences identified in various protein hydrolysates (C). Detailed Implementation

[0073] The terms “peak” and “line” can be interpreted in the same manner and are interchangeable in this disclosure. Similarly, the terms “intensity” and “amplitude” can be interpreted in the same manner and are interchangeable in this disclosure.

[0074] Figure 1 A flowchart is shown for a method for non-targeted identification of peptide sequences from a sample.

[0075] In this document, the term "sample" should be understood to mean a representative portion of a larger population selected for observation, measurement, or analysis purposes in order to draw conclusions about the original population. In the context of this invention, a sample contains several peptides, which may have different amino acid sequence lengths, and for a given amino acid sequence length, may have different amino acid sequences.

[0076] Peptides can be natural peptides and / or synthetic peptides.

[0077] A peptide is referred to as a "natural peptide" when the sample is a portion derived from a natural source or a portion of the product of one or more chemical or biochemical reactions applied to a natural source. In one or more embodiments, the sample contains a natural peptide.

[0078] A peptide is referred to as a "synthetic peptide" when the sample is a portion of a group of peptides artificially produced in the laboratory using methods such as solid-phase peptide synthesis (SPPS), liquid-phase peptide synthesis, or enzymatic synthesis. In one or more embodiments, the sample contains synthetic peptides.

[0079] In one or more embodiments, the sample containing the natural peptide is a protein hydrolysate.

[0080] In this document, the term "protein hydrolysate" or simply "hydrolysate" refers to a product derived from the breakdown of proteins into smaller peptides or even amino acids through a process known as hydrolysis. Hydrolysis is a chemical reaction involving the addition of water to break the peptide bonds between the constitutive amino acids in a protein. This protein fragmentation process results in smaller molecules that are more easily digested and absorbed by the body. The purpose of producing protein hydrolysates is often to obtain products that can be rapidly absorbed by the body, as the peptides and amino acids produced by hydrolysis are generally more soluble and easier to absorb than whole proteins. These hydrolysates are commonly used in the food industry, dietary supplements, and pharmaceutical preparations, particularly for individuals with specific requirements for protein digestion or absorption. The hydrolysates can also be used in the cosmetics industry. Protein hydrolysis can be carried out using various methods, which generally involve the use of chemical agents, enzymes, or a combination of both. The main hydrolysis methods used in the industry, particularly in the food industry, are acid hydrolysis and enzymatic hydrolysis. Hydrolysates can be prepared from a variety of sources, preferably natural sources. Thus, hydrolysates can be derived from microbial proteins, plant proteins, or animal proteins. Protein hydrolysates can be, for example, protein hydrolysates derived from yeast or other microorganisms, whey hydrolysates, casein hydrolysates, soybean hydrolysates, fish protein hydrolysates, collagen hydrolysates, wheat protein hydrolysates, or rice protein hydrolysates.

[0081] In one or more embodiments, the synthetic peptide sample is obtained by chemical synthesis, preferably by solid-phase synthesis (SPPS) or liquid-phase synthesis.

[0082] In one or more embodiments, one or more peptides present in the sample are biologically active.

[0083] In this document, the term "small bioactive peptide" should be understood to mean small peptides that exhibit specific biological activities. These peptides may be of natural or synthetic origin and are capable of modulating a variety of biological processes when they interact with receptors, enzymes, or other target molecules in living organisms. For example, these peptides may exhibit antimicrobial, anti-inflammatory, or antioxidant properties (such as glutathione tripeptide).

[0084] The peptides identified by the method of the present invention are advantageously short peptides (or in other words, small peptides) having 2 to 6 amino acids, typically 2 to 5 amino acids, or even 2 to 4 amino acids. Therefore, the peptides identified by the method of the present invention can be selected from dipeptides, tripeptides, tetrapeptides, pentapeptides, and hexapeptides, as well as mixtures thereof.

[0085] In one or more embodiments, the sample contains a short peptide having 2 to 6 amino acids, wherein at least one end of the short peptide is labeled.

[0086] Preferably, the sample contains a large number of short peptides with different sequences; preferably, the sample contains at least 100 short peptides with different sequences, more preferably at least 200 short peptides with different sequences, even more preferably at least 300 short peptides with different sequences, or most preferably at least 500 short peptides with different sequences.

[0087] In some embodiments, the sample contains up to 10,000 short peptides with different sequences, preferably up to 5,000 short peptides with different sequences, more preferably up to 2,000 short peptides with different sequences, or even more preferably up to 1,000 short peptides with different sequences.

[0088] In some embodiments, the sample contains 100 to 2,000 short peptides with different sequences, preferably 100 to 1,500 short peptides with different sequences, or more preferably 100 to 1,000 short peptides with different sequences.

[0089] In some embodiments, the sample contains 150 to 2,000 short peptides with different sequences, preferably 200 to 2,000 short peptides with different sequences, more preferably 250 to 2,000 short peptides with different sequences, even more preferably 150 to 1,000 short peptides with different sequences, even more preferably 200 to 1,000 short peptides with different sequences, or most preferably 250 to 1,000 short peptides with different sequences.

[0090] Soybean hydrolysate typically contains about 150 short peptides with different sequences.

[0091] Casein hydrolysate typically contains about 300 peptides.

[0092] Yeast hydrolysate typically contains about 500 peptides.

[0093] As detailed below, peptides present in a sample may contain modified amino acids, regardless of the source and nature of the modification. In particular, modified amino acids may have modified side chains.

[0094] In one or more embodiments, for example, the terminal amino acid of one or more peptides in the sample may be labeled at the end. N End and / or C The end. Preferably, the end of the mark is N End.

[0095] Identification methods The method is preferably a non-targeted identification method.

[0096] The term "non-targeted" in relation to identification methods means that the method is performed without any preconceived assumptions about the sequences and amounts of the various peptides contained in the sample. The nature and amounts of the various peptides contained in the sample are unknown beforehand. In other words, the peptide composition and sequences of the peptides in the sample are unknown beforehand.

[0097] The method can be implemented by a device including a memory and a processor.

[0098] The memory may contain at least one predetermined peptide library, the predetermined peptide containing 2 to 6 amino acids, typically 2 to 5 amino acids, or even 2 to 4 amino acids.

[0099] Each predetermined peptide can be defined by a peptide sequence, and for each predetermined peptide, the library can contain: The constitutive amino acids in the peptide sequence and the total mass-to-charge ratio of the peptide sequence ( m / z The peptide sequence has at least one terminal amino acid, and the at least one terminal amino acid is labeled with a terminal tag. Fragment ions calculated based on the peptide sequence, wherein each calculated fragment ion is associated with a mass-to-charge ratio, and at least one or more calculated fragment ions contain the terminal label.

[0100] The total mass-to-charge ratio of the predetermined peptide ( m / z The mass-to-charge ratio of a peptide can be determined by the amino acid sequence of a predetermined peptide. In fact, the sum of the mass-to-charge ratios of all amino acid residues in the peptide sequence of the library can correspond to the total mass-to-charge ratio of the predetermined peptide defined by the peptide sequence.

[0101] In one or more embodiments, fragment ions (or a set of fragment ions) can be calculated based on a peptide sequence by applying a fragmentation method to the peptide sequence.

[0102] It should be noted that, in one or more embodiments, the library may be divided into two or more different libraries. For example, the first library may contain the constitutive amino acids of the peptide sequence and the total mass-to-charge ratio of the peptide sequence (MTB). m / z The peptide sequence has at least one terminal amino acid, which is terminally labeled; and the second library may contain predetermined peptides from the first library, the total mass-to-charge ratio of the predetermined peptides, and fragment ions calculated based on the peptide sequence for each predetermined peptide, the fragment ions being associated with the mass-to-charge ratio, and at least one or more of the calculated fragment ions containing terminal labels. Therefore, this second library can be determined based on the first library. In this configuration, two or more libraries may be used according to the method of this application.

[0103] In this document, "peptide sequence," "amino acid sequence," or "amino acid residue sequence" refers to a description of the arrangement of the constitutive amino acids (monomers) in a peptide. In fact, a peptide is an ordered linear polymer composed of amino acids. Sequences are typically represented as strings stored in text format in a computer file.

[0104] In this document, "amino acid" refers to a residue containing a central carbon atom (α-carbon) bonded to a carboxyl group (-COOH), an amino group (-NH2), a hydrogen atom (-H), and a side chain (denoted as -R). This side chain (also called a side chain group) is what distinguishes amino acids from one another. In all cases except glycine, the central α-carbon is asymmetric (because glycine has a hydrogen atom instead of a side chain). The same amino acid can have two different configurations, which cannot be interconverted without breaking and reforming covalent bonds. These configurations are designated L and D. Although both can be synthesized in the same chemical reaction, only the L form is used for protein synthesis (protein-derived), while the non-protein-derived D form is incorporated into the synthesis of carbohydrates or lipids in living organisms. In particular, amino acids can be protein-derived amino acids or modified amino acids.

[0105] The term "protein-derived amino acids" or "unmodified amino acids" refers to amino acids that serve as the basic building blocks of proteins. Protein-derived amino acids are the basic building blocks of proteins. They polymerize to form linear polypeptides, in which amino acid residues are linked by peptide bonds. Protein biosynthesis occurs on ribosomes, which translate messenger RNA into proteins. The order in which amino acids are linked in a polypeptide chain is specified by the sequence of codons carried by a messenger RNA sequence, which is a copy of DNA from the cell nucleus. These codons, as nucleotide triplets, are translated into amino acids via transfer RNA according to the genetic code. This code directly specifies 20 standard protein-derived amino acids (A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V). Two additional amino acids were added to the code via more complex mechanisms: for selenocysteine ​​(U), the more complex mechanism involved recoding the SECIS element of the UGA stop codon, and for pyrrolysine (O), the more complex mechanism involved recoding the PYLIS element of the UAG stop codon. Thus, a total of 22 protein-derived amino acids exist. Pyrrolysine is found only in proteins of certain methanogenic archaea; therefore, eukaryotes and bacteria utilize only 21 protein-derived amino acids.

[0106] The most common amino acids are represented by 1-letter or 3-letter codes, as shown in the table below. When a particular amino acid is not fully identified (a vague amino acid), the symbols B, Z, and J are used.

[0107]

[0108] [Table 1]: Amino Acids: Symbols, Three-Letter Codes, and Definitions In this document, the term “peptide” can be used interchangeably to refer to a peptide present in a sample (in other words, the peptide being tested) or a peptide in a library (in other words, the predetermined peptide).

[0109] In one or more embodiments, the peptide contains amino acids selected from protein-derived amino acids and modified amino acids.

[0110] A peptide contains at least one terminal amino acid with a terminal label.

[0111] In one or more embodiments, the peptide contains at least one labeled terminal amino acid and one or more amino acids selected from protein-derived amino acids and modified amino acids.

[0112] In one or more embodiments, the labeled terminal amino acid may be a protein-derived amino acid or a modified amino acid. For example, the labeled terminal amino acid may be an amino acid with a modified side chain.

[0113] "Modified amino acids" is understood to refer to any non-protein-derived amino acid. Modified amino acids can be products of endogenous or exogenous chemical modifications applied to protein-derived amino acids. Modified amino acids can also be synthetic amino acids.

[0114] In this document, endogenous reactions are reactions that occur naturally within an organism or biological system, such as post-translational modification reactions, while exogenous reactions are reactions triggered or caused by factors outside the organism or biological system. Examples of exogenous reactions include peptide oxidation (e.g., chemical or photochemical oxidation) that occurs outside of a biological organism under laboratory or industrial conditions, or protective reactions involving protecting groups, such as those to protect amino acids from oxidation.

[0115] In one or more embodiments, the modified amino acid is an amino acid in which the side chain, carboxyl group, or amino group is modified.

[0116] In one or more embodiments, the modified amino acid is an amino acid containing a modified side chain. The modified side chain can be, for example, a product of endogenous chemical modification (such as post-translational modification), or a product of one or more exogenous chemical or biochemical reactions, such as a product of reaction with a side chain protecting group. The modified amino acid can also be a product of a reaction in which atoms in the side chain are replaced with equivalent atoms (e.g., a fluorine atom replaces a hydrogen atom, a selenium atom replaces a sulfur atom, or a phosphorus atom replaces a nitrogen atom).

[0117] Regardless of its origin, any chemical modification of an amino acid (especially its side chain) can be detected by the method of the present invention, provided that the amino acid is included in the library.

[0118] Examples of modifications to the side chains of cysteine, lysine, and methionine are detailed below.

[0119] Cysteine ​​is an amino acid containing a thiol group (-SH), which makes it reactive. It may be necessary to protect cysteine ​​from oxidation or other undesirable reactions. Protecting cysteine ​​typically involves forming derivatives that make it less reactive.

[0120] In some embodiments, the amino acid containing the modified side chain is cysteine, whose side chain has been chemically modified to render it non-reactive. For example, cysteine ​​can be carbamoylmethylated. Carbamoylmethylation of cysteine ​​can be performed by contacting the sample with an alkylating agent such as iodoacetamide, which is typically used to protect the free sulfhydryl groups of cysteine ​​from oxidation. Cysteine ​​can be used with... N-Other types of protecting groups, such as ethyl maleimide, protect (Schilling, D., Barayeu, U., Steimbach, RR, Talwar, D., Miller, AK and Dick, TP (2022), Commonly used alkylating agents limit persulfide detection by converting protein persulfides into thioethers. Angewandte Chemie, 134(30), e202203684. DOI: 10.1002 / anie.202203684. PMID: 35506673).

[0121] In some embodiments, the amino acid containing the modified side chain is a lysine with an acetylated side chain, typically acetylated lysine. Acetyllysine can be endogenously formed in certain proteins through post-translational modification of lysine residues. Therefore, this amino acid can be present in the peptide of the sample, even if it is not a protein-derived amino acid.

[0122] In some embodiments, the amino acid containing the modified side chain is methionine with an oxidized side chain, typically methionine sulfoxide.

[0123] Methionine sulfoxides can be formed in certain proteins through post-translational modification of methionine residues. Therefore, this amino acid can be present in the peptide of a sample, but it is not a protein-derived amino acid.

[0124] In some embodiments, the amino acid is a selenium-containing amino acid. Selenium-containing amino acids are analogs of standard amino acids that contain selenium, rather than sulfur, in their structure. These selenium-containing amino acids can be incorporated into proteins during protein biosynthesis to replace their corresponding sulfur-containing amino acids. The main selenium-containing amino acids are selenomethionine (Sem) and selenocysteine ​​(Sec), where Sem and Sec are selenium equivalents of methionine and cysteine, respectively. Peptides containing selenium-containing amino acids can be prepared by chemical peptide synthesis, recombinant expression, biocatalysis, or post-synthetic modification techniques.

[0125] Importantly for the method of this invention, the peptide contains at least one terminal amino acid with a terminal label. The terminally labeled amino acid may be in which an amino group (-NH2) or a carboxyl group (-COOH) is modified to label its respective constituents. N end or CThe amino acids of the terminal peptide.

[0126] In this document, "terminal label" is understood to mean a label specifically used to label proteins or peptides. N end or C A pharmaceutical or chemical group labeled at or on the end of the substance. Therefore, in N End markers and C Distinguishing between terminal markers. These terminal markers are known in molecular biology research, proteomics, and protein identification, purification, and analysis.

[0127] In one or more embodiments, amino acids are N Terminal amino acids, in which the amino group (-NH2) is... N End marker modification.

[0128] N The terminal label may be specifically selected from dansyl, dabusulfonyl, 1-naphthyl isocyanate (IsoC) or 1-naphthyl isothiocyanate, wherein each of these terminal labels is optionally carbon-13 ( 13 C) Deuterium (D) Nitrogen-15 ( 15 N) and / or sulfur-34 ( 34 One or more atomic tags in S).

[0129] Optional use of carbon-13 ( 13 C) Deuterium (D) Nitrogen-15 ( 15 N) and / or sulfur-34 ( 34 One or more atomically labeled dansyl groups in S) are preferred.

[0130] In one or more embodiments, the terminal amino acid is wherein the carboxyl group (-COOH) is... C End marker modification C Terminal amino acids.

[0131] C The terminal marker can be specifically selected from 2-( N,N' -dimethylamino)ethyl-1-amine (DMED) or 3-( N,N' -dimethylamino)prop-1-amine (DMAPA), wherein each of these terminal tags is optionally represented by carbon-13 ( 13 C), deuterium (D) and / or nitrogen-15 ( 15 One or more atomic labels in N).

[0132] In one or more embodiments, the peptides present in the sample are peptides in which only one terminal amino acid is terminally labeled; in other words, they are peptides in which only one terminal end is terminally labeled.

[0133] In one or more embodiments, the peptide is a peptide in which both terminal amino acids are terminally labeled; in other words, they are peptides with both ends labeled.

[0134] A library can be calculated (or generated) such that a predetermined peptide contains amino acids selected from: protein-derived amino acids, modified amino acids (especially amino acids containing modified side chains), and mixtures thereof, provided that the predetermined peptide contains at least one terminal amino acid with a terminal label.

[0135] One or more predetermined peptide libraries may be generated by the apparatus implementing the method prior to each sample measurement, or may be received by any means of communication. For example, the library may be generated on a third-party device and transferred from said device to the apparatus implementing the method.

[0136] In one or more embodiments, the predetermined peptide library may contain all possible peptide sequences consisting of 2 to 6 amino acids, said amino acids being obtained from a set of N amino acids.

[0137] The term “all possible peptide sequences” should be understood to mean all possible combinations of peptide sequences that form a length of 2 to 6 amino acids (e.g., 2 to 4 amino acids), said amino acids being obtained from a set of N amino acids.

[0138] For example, N can be 2 to 50 amino acids. Only about twenty natural amino acids are present. However, as detailed below, libraries can be constructed to account for the presence of modified amino acids in the test peptide, whether these modified amino acids are of natural or synthetic origin. Therefore, in one or more embodiments, N can preferably comprise at least 22 protein-derived amino acids and some modified versions of these protein-derived amino acids. In one or more embodiments, N can be greater than 2, preferably greater than 20. In one or more embodiments, N can be less than 500, preferably less than 400, more preferably less than 300, or even more preferably less than 200. N can typically be between 2 and 100, for example, between 2 and 50.

[0139] Therefore, each sequence in the predetermined library can define a predetermined peptide in the library, and can further include tagged terminal amino acids.

[0140] Furthermore, in one or more embodiments, the memory may contain information sets associated with peptides (referred to as analytes) present in the sample, measured by a mass spectrometer, each analyte potentially containing a peptide sequence having at least one terminal amino acid with a terminal label. Each information set of the analytes may include at least one MS / MS spectrum containing the total mass-to-charge ratio of the analyte and one or more mass-to-charge ratios of the analyte fragment ions.

[0141] Each fragment ion can represent one or more linked amino acids (or amino acid residues) within the peptide sequence of the peptide being tested.

[0142] In addition, according to one or more embodiments, each information set associated with the analyte peptide may also include mass spectrometry (referred to as MS spectrum) information, wherein the MS spectrum information may include the mass-to-charge ratio, retention time, and intensity of the analyte peptide (i.e., the precursor ion) of the analyte peptide (i.e., the precursor ion).

[0143] In addition, the peptide to be tested and at least a variety of predetermined peptides from a predetermined peptide library can be labeled with the same end label at the same end.

[0144] According to one or more examples, the mass spectrometer may be a mass spectrometer (MS / MS) with tandem analysis capability, preferably coupled with liquid chromatography (LC), such as a mass spectrometer using a liquid chromatography column.

[0145] This mass spectrometer, optionally coupled with liquid chromatography, allows for the acquisition of a set of information about the analyte peptide in the sample, such as the mass spectrometry (MS) and / or MS / MS fragmentation spectra of the peptide (also known as the precursor ion), thereby providing information about the structure of the peptide (or precursor ion) based on the fragment ions present in the MS / MS spectrum.

[0146] For example, the information set associated with these spectra (MS and MS / MS) may include the mass-to-charge ratio and peak intensity corresponding to the constitutive fragment ions and / or parent ions in the analyte peptide, as well as the retention time of the analyte peptide.

[0147] Furthermore, the information set of the peptide being tested can include an MS spectrum and one or more MS / MS spectra, the contents of which are usually similar. In such cases, it can be decided whether to use a single MS / MS spectrum (e.g., the MS / MS spectrum with the highest intensity precursor ion) or multiple MS / MS spectra.

[0148] Liquid chromatography (LC), using, for example, a chromatographic column, can be used to separate peptides, which can then be measured by a mass spectrometer, and retention time can also be determined.

[0149] This retention time refers to the length of time a peptide from a sample remains in the chromatographic column (of the chromatograph) before being eluted (i.e., until the peptide arrives at the mass spectrometer). Therefore, this retention time (for each peptide) can be measured from the moment the sample is injected into the chromatograph until the moment the peptide arrives at the mass spectrometer.

[0150] Figure 2 An example of a pre-defined peptide library is illustrated.

[0151] In this illustrative example, the resulting library may contain all possible peptide sequences (SEQ_PEP) (twelve in the case shown), each peptide sequence containing two to three amino acids, which may be obtained from a set of N = 3 amino acids (A, B, C), and each peptide sequence in the library may contain a terminal amino acid with a terminal label.

[0152] As mentioned above regarding peptides in the sample, the terminal amino acid markers of the peptide sequences in the library can be... N End and / or C The end. Preferably, the end of the mark is N Terminal (or N-terminal type).

[0153] For example, in Figure 2 In the case of a library, the end of the tag is N The terminus. Therefore, the predetermined peptide PP2 is marked with... The terminal amino acid of the terminal label is amino acid B, and the predetermined peptide PP8 is labeled with amino acid B. The terminal amino acid of the terminal label is amino acid A, and so on.

[0154] Each peptide sequence (SEQ_PEP) can define the corresponding predetermined peptide PP1...PP12 in the library, and can be used to determine or calculate the total mass-to-charge ratio of the predetermined peptide. m / z m1……m12, as described above (R_seq_pep). For example, mass-to-charge ratio ( m / z m7 can be derived from the peptide sequence A of peptide PP7. -BC calculation (e.g., by summation), where A It is the labeled terminal amino acid (by summing the mass-to-charge ratio of the constitutive amino acid residues in the peptide sequence).

[0155] It should also be noted that, Figure 2 In the example, the ratios m1 and m2 are equal because the peptide sequences of the predetermined peptides PP1 and PP2 contain the same number of the same amino acids. This reasoning can also be applied to predetermined peptides PP3 and PP4, predetermined peptides PP5 and PP6, and predetermined peptides PP7 through PP12.

[0156] It should be noted that the mass-to-charge ratio of the labeled terminal amino acids takes into account the terminal label. In fact, the mass-to-charge ratio of amino acid A is the same as that of the amino acid labeled with terminal label. Terminally labeled amino acid A Their mass-to-charge ratios are different.

[0157] Of course, in Figure 2 In the example, the end of the tag is N Terminal (or N-terminal type), but it can also be C Terminal (or C-terminal type). For example, a pre-defined library may contain documents with... N Predetermined peptides with terminal tags and C The predetermined peptide with terminal labeling, both and / or with C End markers and N Predetermined peptides with terminal labels.

[0158] According to one or more examples, the test peptide and the predetermined peptide in the predetermined peptide library can be labeled with the same end tag at the same end.

[0159] For example, the peptide being tested can contain all of the following: N Terminal (or N-terminal) tags, and the predetermined peptide library contains at least a plurality of peptides with terminal (or N-terminal) tags. N The terminal label is selected from the following predetermined peptides: having N End markings and / or C A predetermined peptide with terminal labeling and / or a combination of both.

[0160] In one or more embodiments, the peptide sequences in the library may all have the same end label, for example... N end or C End.

[0161] In one or more embodiments, the amino acids in a set of N amino acids may be protein-derived amino acids or modified amino acids. For example, modified amino acids may be amino acids having modified side chains.

[0162] Therefore, the predetermined peptide can contain labeled N end or C Terminal amino acids, protein-derived amino acids, and amino acids with modified side chains. Preferably, the amino acids with modified side chains are selected from the group consisting of cysteine, lysine, and methionine.

[0163] According to one or more embodiments, the cysteine ​​of the predetermined peptide is a cysteine ​​with a modified side chain (preferably modified by carbamoyl methylation). This modification is specifically used to prevent the unwanted formation of disulfide bonds during sample preparation and analysis.

[0164] Furthermore, according to one or more examples, the library may contain at least one predetermined peptide in the library, the at least one predetermined peptide being represented as a plurality of charged predetermined peptides, each charged predetermined peptide having the same peptide sequence as the at least one predetermined peptide and a corresponding charge state.

[0165] "A pre-charged peptide" can be understood as a pre-charged peptide having one or more positive or negative charge states.

[0166] Thus, for example, a large number of charged versions can be derived from one or more predetermined peptides in the library, each charged version exhibiting its own charge state (or corresponding charge state) but sharing the same peptide sequence as the predetermined peptide from which it is derived.

[0167] Advantageously, the peptide sequence of the test peptide carrying one or more charges (e.g., single-charged, double-charged, or triple-charged) can thus be determined.

[0168] exist Figure 2 The document also demonstrates fragment ions (or a set of fragment ions) that can be calculated based on the amino acid sequence of a predetermined peptide. Each fragment ion (or peptide fragment ion or fragment) in the fragment ion (Ions_Frag) can be associated with a corresponding mass-to-charge ratio (R_ion_frag).

[0169] For example, the predetermined peptide PP1 may contain the peptide sequence (Seq-pep) A. - B, and fragment ions (or a set of fragment ions) of the sequence A B and from sequence A - Other fragments calculated (or obtained) by B (through the fragmentation method), where A It is with Terminally labeled terminal amino acids. The fragment ions (or a set of fragment ions) calculated for a predetermined peptide PP2 may include fragment ion B. Fragment ion A and sequence B Other fragments of -A, including fragment ion B Include End marker.

[0170] According to one or more examples, the fragmentation method may be the same as that of a mass spectrometer or a mass spectrometer (MS / MS) with tandem analysis capability (or a system that couples tandem mass spectrometry with liquid chromatography).

[0171] In fact, fragment ions obtained from (or calculated from) the corresponding peptide sequence in a predetermined peptide library can be equivalent to peptides (or parent ions) that are fragmented by tandem mass spectrometry to produce the fragment ions to be measured.

[0172] Based on an example, the fragmentation method could be: - Collision-induced dissociation (CID) - Electron transfer dissociation (ETD) - Electron capture and dissociation (ECD) - Electron collisional dissociation (EID) - Infrared multiphoton dissociation (IRMPD) - UV photodissociation (UVPD).

[0173] Furthermore, based on one or more examples, fragment ions can be calculated from a predetermined peptide by applying various fragmentation methods.

[0174] For example, each fragmentation method can be specific to the type of mass spectrometer (or tandem mass spectrometry system) using that particular fragmentation method. As one example, in a predetermined peptide library, peptide sequences can contain fragments (or fragment ions), such as fragments grouped into multiple sets of fragment ions, where the first set of fragment ions is obtained using a collision-induced dissociation (CID) fragmentation method, and the second set of fragment ions can be obtained using an electron transfer dissociation method (see the list of examples above).

[0175] Therefore, the same library can be advantageously used to identify peptides in a sample that have been measured by different mass spectrometers (or tandem mass spectrometry systems), each using a specific fragmentation method or capable of using different fragmentation methods (see the list above).

[0176] refer to Figure 1 The method may include using a library and the at least one MS / MS spectrum to determine the selection of 110 predetermined peptides.

[0177] The total mass-to-charge ratio of each selected predetermined peptide can be equal to the total mass-to-charge ratio of the peptide being tested, with an error tolerance lower than a predetermined value.

[0178] Therefore, for example, determining the selection of the test peptide can consist of comparing the total mass-to-charge ratio of the test peptide (i.e., the mass-to-charge ratio of the parent ion in the MS / MS spectrum of the test peptide) with the total mass-to-charge ratio of each predetermined peptide present in the library, and selecting a predetermined peptide from the library whenever the total mass-to-charge ratio present in the library is equal to (or close to) the total mass-to-charge ratio of the test peptide (the mass-to-charge ratio of the parent ion) and the error tolerance is below a predetermined value.

[0179] Based on an example, and referring to Figure 2 If the mass-to-charge ratio of the peptide being tested is equal to or close to ratio m1, then select ( Figure 2The shaded area in the diagram will include the predetermined peptide PP1, but also the predetermined peptide PP2, because the ratio m2 equals the ratio m1. In fact, the predetermined peptides PP1 and PP2 contain the same amino acids, the same number of amino acids, and the same terminal label.

[0180] According to one or more examples, the predetermined value of the error tolerance may be less than 20 ppm (parts per million), preferably less than 15 ppm, more preferably less than 10 ppm, and even more preferably less than 5 ppm.

[0181] After determining the selection of the analyte peptide, the method may include, while maintaining an error tolerance below a predetermined value, comparing the mass-to-charge ratio of the analyte fragment ions with the calculated fragment ion mass-to-charge ratio of the selected predetermined peptide (i.e., the mass-to-charge ratio of the fragment ions). Figure 2 The mass-to-charge ratio of fragment ions in the shaded region of the example was compared to determine the terminal amino acid of the labeled peptide of 120.

[0182] According to one or more examples, the comparison may involve the mass-to-charge ratio of the analyte fragment ion with terminal labeling (detected in the MS / MS spectrum of the analyte peptide), which is compared with the mass-to-charge ratio of selected fragment ions and / or the mass-to-charge ratio of selected fragment ions with terminal labeling.

[0183] Therefore, identifying the labeled terminal amino acid before any sequence reconstruction makes it possible to determine the terminal amino acid of the sequence, which can then be used as a starting point for reconstructing the peptide sequence of the peptide being tested.

[0184] Then, the method can include fragment ions calculated based on the selection (i.e., Figure 2 The method uses fragment ions (in the shaded portion of the example), the identified labeled terminal amino acids, and the analyte fragment ions to determine at least one reconstructed peptide sequence of 130 analyte peptides. The method may then include delivering an identification list of 140 peptides that identify the analyte peptides in the sample, wherein each analyte peptide in the list is defined by a reconstructed peptide sequence.

[0185] According to one or more examples, each test peptide in the list can be associated with an information set (e.g., MS / MS spectrum) of the reconstructed peptide sequence used to identify the test peptide by means of identification. For example, the identification means can correspond to numbering each information set, and each test peptide in the list can be associated with a corresponding number that corresponds to the number of the information set of the reconstructed peptide sequence used to identify the test peptide.

[0186] It should be noted that when using multiple libraries (such as the two libraries described above), the first library can be used to determine the selection, and the second library can be used to reconstruct the sequence, wherein the predetermined peptides of the first library (and thus the selected peptides) are also present in the second library.

[0187] According to one or more examples, preferably, the first library as described above can also be used and a second library containing only the selected predetermined peptides (as well as the calculated fragment ions and the calculated mass-to-charge ratio of the fragment ions) can be calculated as described above.

[0188] According to one or more embodiments, the peptide sequence of the tested peptide (i.e., at least one peptide sequence) can therefore be reconstructed based on the following: - The identified terminal amino acid of the label, - Comparison between the mass-to-charge ratio of the measured fragment ions and the calculated mass-to-charge ratio of the fragment ions.

[0189] During the reconstruction of the peptide sequence of the test peptide, i.e., after determining the labeled terminal amino acid (starting point), the reconstructed peptide sequence of the test peptide is determined by comparing the test fragment ions with calculated fragment ions (from the selection). For example, the comparison may involve various mass-to-charge ratios of available fragment ions (meaning the test fragment ions and / or calculated fragment ions).

[0190] In one or more embodiments, the peptide sequence of each tested peptide can be reconstructed using methods derived from those used in so-called "de novo" sequencing. In short, "de novo" sequence reconstruction is based on assembling overlapping fragmented sequences to derive the complete sequence. Reconstruction methods can be based on the following articles: - Biemann, K. (1992). Mass spectrometry of peptides and proteins. Annual review of biochemistry, 61(1), 977-1010, DOI: 10.1146 / annurev.bi.61.070192.004553. PMID: 1497328.

[0191] - Wysocki, VH, Resing, KA, Zhang, Q. and Cheng, G. (2005). Mass spectrometry of peptides and proteins. Methods, 35(3), 211-222. DOI: 10.1016 / j.ymeth.2004.08.013. PMID: 15722218.

[0192] - Seidler J, Zinn N, Boehm ME, Lehmann WD. De novo sequencing of peptides by MS / MS. Proteomics. Feb. 2010;10(4):634-49. Doi: 10.1002 / pmic.200900459. PMID: 19953542.

[0193] The peptide sequence of a peptide can be reconstructed using the principle of "de novo" sequencing, based on one or more MS / MS spectra of the peptide to be tested, the identified labeled terminal amino acids, and fragment ions from a selected predetermined peptide.

[0194] During the measurement of peptides by mass spectrometry, the collected data may include irrelevant elements. Such irrelevant data may arise from artifacts produced by the mass spectrometer or from the simultaneous presence of another peptide (different from the peptide being measured). The latter is particularly likely to occur when two peptides are co-eluted in the mass spectrometer with similar retention times.

[0195] Figure 3 This schematically illustrates the co-elution phenomenon of the two peptides.

[0196] It should be noted that co-elution can involve more than two peptides that may have different peptide sequences and / or the same peptide sequence but different charge states, such as three or four peptides or more.

[0197] like Figure 3 As shown, each retention time RT1 and RT2 corresponds to the retention time of the peptide in the sample after it has migrated from the chromatographic column to the mass spectrometer (or tandem mass spectrometry system). Figure 3 As can be seen, in the case of co-elution, the chromatographic peaks of the two peptides may partially or completely overlap, for example, due to their very similar retention times. In this case, the tandem mass spectrometer can then record a set of information, namely the combined spectrum of the co-eluted peptides (also known as the chimeric spectrum).

[0198] This situation can make it difficult to determine the peptide sequence that actually corresponds to the peptide being tested, because the information set of the peptide being tested (e.g., one or more MS / MS spectra) may include multiple peptide sequences, and each peptide sequence may potentially correspond to the peptide sequence of the peptide being tested.

[0199] Therefore, in one or more embodiments, when multiple reconstructed peptide sequences are determined for the same test peptide, for example in the presence of interfering data due to co-elution of one or more peptides, the method may further include: - A score is calculated for each reconstructed peptide sequence based on the information set associated with the peptide being tested and based on the reconstructed peptide sequence. - The reconstructed peptide sequence of the test peptide was selected based on a comparison of the scores of the reconstructed peptide sequences, and the peptide sequence with the highest score was selected.

[0200] The reconstructed peptide sequence with the highest score then corresponds exactly to the peptide being tested and can be delivered (output) in the identification list along with the obtained score. Only the peptide sequence with the highest score is retained in the identification list.

[0201] However, the reconstructed sequence of the analyte peptide with a lower score and corresponding to one or more co-eluted peptides can also correspond to the peptide in the mixture that needs to be measured and identified, for example, because said peptide also has a peptide sequence of 2 to 6 amino acids in length. Therefore, these peptides eluted with the analyte peptide (e.g., having the same peptide sequence but different charge states and / or different peptide sequences) can also correspond to the analyte peptide and can generally be identified before or after the analyte peptide, depending on the time resolution of the mass spectrometer (or tandem mass spectrometry system).

[0202] For example, refer to Figure 3 The peptide sequences of the test peptide PP1 eluted at retention time RT1 (close to retention time RT2) and the test peptide eluted at retention time RT2 can be determined at these two identical retention times. In this case, i.e., in the case of co-elution, the reconstructed sequence with the highest score will correspond to peptide PP1 at retention time RT1, and it will correspond to PP2 at retention time RT2. Both peptide sequences will be included in the identification list at the same retention time.

[0203] In the case of co-elution, an alert can be issued (e.g., to the user or recorded in the identification list), and the identification list can be post-processed (e.g., result verification). The co-elution may result in the presence of multiple analyte peptides with similar or identical retention times in the identification list. For example, retention time data of analyte peptides obtained from various data sources (LC chromatograms, MS spectra, etc.) can be compared to determine whether the retention time truly corresponds to the analyte peptide (e.g., by using extracted ion chromatograms (XIC) detected by MS and comparing peak intensities and / or retention times in MS and MS / MS spectra). In another instance, when multiple peptides share the same sequence but have different charge states, post-processing may consist of, for example, retaining only the peptide with the highest intensity in the list.

[0204] In one or more embodiments, each mass-to-charge ratio of the analyte fragment ion can be correlated with its intensity in the MS / MS spectrum, and the score corresponding to each reconstructed peptide sequence of the same analyte peptide can be determined using the following equation:

[0205] The intensity of fragment ions can also be understood as the intensity of fragment ion peaks.

[0206] Based on one or more examples, a score for a peptide sequence can be calculated using fragment ions with only end labels.

[0207] Therefore, for example, refer to Figure 2 If the fragment ions in the MS / MS spectrum of the peptide being tested correspond to the predetermined peptides PP1 and PP2 in the library (i.e., Figure 2 In the example selection (shaded area), only fragments A of PP1 and PP2 can be used. - A B and B - B A calculates their respective scores.

[0208] In one or more embodiments, one or more labeled terminal amino acids may be identified, and for each identified labeled terminal amino acid, at least one reconstructed peptide sequence of the test peptide may be determined based on the calculated fragment ions selected, the identified labeled terminal amino acid, and the test fragment ions.

[0209] In fact, as described above, in the presence of co-elution of one or more peptides (e.g., peptides with the same sequence but different charge states and / or different sequences), multiple reconstructed peptide sequences can be identified. These reconstructed peptide sequences may have the same identified labeled terminal amino acid or may have different identified labeled terminal amino acids. For example, in peptide sequence A with the same (or very close) mass-to-charge ratio... - B - C and A In C - B, A It is the terminal amino acid of the identified marker shared by both sequences. According to another example, in sequence A with the same (or very close) mass-to-charge ratio... - B - C and B - A - C, A and B These are the terminal amino acids of the identified tags that differ in the two sequences. In the latter case, during peptide sequence reconstruction, each identified terminal amino acid of the identified tags is considered as the starting point of at least one peptide sequence to be reconstructed.

[0210] Furthermore, in one or more embodiments, the list may include retention times associated with the test peptide, each retention time being obtained from the information set of the reconstructed peptide sequence used to determine the test peptide, particularly from, for example, MS spectra (and / or also from MS / MS spectra).

[0211] Each peptide in the list can be associated with a corresponding retention time obtained from the information set (e.g., MS spectrum) of the reconstructed peptide sequence used to determine the peptide.

[0212] According to one or more examples, the retention time of each analyte peptide can be obtained from liquid chromatography (LC) coupled with mass spectrometry detection (such as a tandem mass spectrometry (MS / MS) system).

[0213] As described above, retention time can be obtained from measurements performed by liquid chromatography and transcribed (or indicated) in the MS spectrum of the parent ion (and / or one or more MS / MS spectra derived from the parent ion).

[0214] Depending on one or more instances, the list may further include intensities associated with the test peptides, each obtained from an information set (e.g., an MS spectrum of the test peptide) used to determine the test peptide. Each test peptide in the list may include a proportion value indicating the percentage of the peptide in the mixture; the proportion value "Qp" for each test peptide in the list can be calculated using the following:

[0215] Advantageously, this allows us to obtain values ​​representing the relative proportions of peptides in the mixture.

[0216] Figure 4 An apparatus for implementing the methods of this disclosure is shown.

[0217] In this embodiment, the apparatus 400 may include a processing circuit system 403 and a memory 402 for storing program instructions, which can be loaded into the circuit system and, when executed by the circuit system 403, enable the circuit system 403 to perform the methods of this disclosure.

[0218] The memory 402 may also store data and information that can be used to perform the methods described in this specification as described above.

[0219] The processing circuit system 403 may be, for example: - A processor or processing unit capable of interpreting instructions in a computer language, wherein the processor or processing unit may include, be associated with or attached to, a memory containing the instructions, or - The association between a processor / processing unit and memory, wherein the processor or processing unit is adapted to interpret instructions in a computer language, and the memory contains the instructions, or - A circuit board in which the sequence of methods is hard-coded in its circuit system, or - Programmable electronic chips, such as FPGAs (Field Programmable Gate Arrays). - GPU (Graphics Processing Unit).

[0220] This device may include an input interface 405 for receiving input data and an output interface 407 for providing useful datasets and / or for controlling a characterization system (e.g., a tandem mass spectrometry system coupled with liquid chromatography).

[0221] For example, input interface 405 can receive input data, such as a predetermined peptide library or multiple predetermined peptide libraries (e.g., those described above), or parameters used to generate the predetermined peptide libraries, or information sets associated with peptides present in the sample as measured by a mass spectrometer. Additionally, optionally, input interface 405 can be connected to a mass spectrometer, such as a tandem mass spectrometry system (a mass spectrometer (MS / MS) coupled with liquid chromatography), to allow measurement of the sample and direct reception from the mass spectrometer of information sets associated with peptides present in the sample.

[0222] For example, output interface 407 can provide, for instance, a generated predetermined peptide library or multiple predetermined peptide libraries (such as those described above) or an identification list identifying the analyte peptides in the sample. Additionally, optionally, the output interface can be connected to a mass spectrometer, such as a tandem mass spectrometry system coupled with liquid chromatography, to allow, for example, control of the mass spectrometer, or direct access from the mass spectrometer to a set of information relating to the peptides present in the sample, for example, sending said information set to a third-party device, such as a computer.

[0223] According to one example, device 400 may be a computer 401 including a processing circuit system 403 and a memory 402. According to another example, the device may be a computer for a mass spectrometer. The computer for a mass spectrometer (MS or LC-MS / MS) may be used, for example, to control the mass spectrometer to measure samples, generate or receive one or more libraries, or implement the methods of this disclosure.

[0224] To facilitate interaction with device 400 or computer 401, a screen 411, a keyboard 412, and a mouse 413 may be provided, and the screen, the keyboard, and the mouse may be connected to the processing circuitry system 403.

[0225] Analytical methods This disclosure also relates to a method for analyzing peptides having 2 to 6 amino acids from a sample, the method comprising: i) A sample processing step, the sample processing step comprising at least one step i1) of labeling the ends of the peptides in the sample by contacting the sample with an end label. ii) Analytical steps, wherein the analytical steps employ liquid chromatography coupled to a mass spectrometer, preferably a mass spectrometer capable of tandem mass spectrometry (MS / MS). iii) A data processing step, wherein the data processing step implements the method for non-targeted identification of peptide sequences as described herein.

[0226] Peptides, amino acids, terminal labels, and the samples analyzed are as described above.

[0227] In one or more embodiments, the peptide is a natural peptide and / or a synthetic peptide.

[0228] In one or more embodiments, the analyzed peptide has 2 to 4 amino acids.

[0229] In one or more embodiments, the peptide undergoing step ii) contains at least one labeled terminal amino acid and one or more amino acids selected from protein-derived amino acids and modified amino acids.

[0230] In one or more embodiments, the sample is a mixture of natural small peptides or a mixture of peptides obtained through peptide synthesis. The sample is preferably a protein hydrolysate, such as yeast protein hydrolysate, plant protein hydrolysate, or animal protein hydrolysate.

[0231] In one or more embodiments, sample processing step i) further includes step i2) of alkylating the peptide by contacting the peptide of the sample with an alkylating agent, and step i2) is preferably prior to step i1).

[0232] This optional alkylation step is performed to protect amino acids with free thiol groups (-SH), such as cysteine, from unwanted chemical reactions, such as oxidation that may occur during subsequent steps of the process.

[0233] Those skilled in the art know how to select a compatible liquid chromatography technique for coupling with mass spectrometry.

[0234] In some embodiments, the liquid chromatography is reversed-phase liquid chromatography, preferably performed using a gradient of at least one hour.

[0235] In some embodiments, the liquid chromatography (LC) employed is nano-LC or micro-LC.

[0236] Nano LC is a high-performance liquid chromatography (HPLC) technique used to separate and analyze samples at flow rates of hundreds of nanoliters per minute (nL / min). It shares similar principles with conventional liquid chromatography but operates at much lower flow rates and sample volumes, typically at the nanoliter scale.

[0237] Micro LC, also known as micrometer-scale liquid chromatography or capillary LC, is a liquid chromatography (LC) technique that uses smaller columns and reduced flow rates ranging from hundreds of microliters per minute (μL / min) compared to conventional liquid chromatography (HPLC). In terms of flow rate and column size, Micro LC falls between standard HPLC and Nano LC.

[0238] In some embodiments, the mass spectrometer is equipped with a mass analyzer, preferably selected from quadrupole ion trap mass analyzers and triple quadrupole mass analyzers; more preferably, the mass spectrometer is equipped with a high-resolution mass analyzer, selected from orbital trap mass analyzers, time-of-flight (Q-TOF) mass analyzers, TIMS-TOF (trapped ion mobility spectrometry-time-of-flight) mass analyzers, and Fourier transform ion cyclotron resonance (FT-ICR) mass analyzers.

[0239] This disclosure is illustrated in a non-limiting manner by way of the following examples.

[0240] Example Example 1: A method for detecting short peptides in protein hydrolysates Purpose In this example, the aim is to characterize short peptides present in yeast extract obtained through the hydrolysis of yeast extract. During hydrolysis, proteins in the yeast extract are broken down into peptides that can be broken down as follows: - Oligopeptides containing 5 to 35 amino acids, - Short peptides with 2 to 5 amino acids, - Free amino acids.

[0241] Short peptides and free amino acids constitute the dominant components. The proteins and oligopeptides can be analyzed using conventional analyses in proteomics and peptidomics. In the first step, the peptide mixture is fractionated using chromatographic techniques (particularly liquid chromatography) or capillary electrophoresis; the peptides are detected and then fragmented in a mass spectrometer to obtain amino acid sequence information. Data are typically obtained by liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS). The MS and MS / MS data are then processed using software to search for mass in a protein data catalog. m / zPeptides are identified by sequence homology and other methods. However, these techniques are unreliable for small peptides, which is why most software programs do not allow the analysis of peptides containing fewer than 5 or 6 amino acids.

[0242] This paper presents a method for analyzing the protein content of hydrolyzed yeast extracts. Short peptides, the dominant components of yeast extracts, have been found to pose challenges in LC-MS / MS analysis related to their physicochemical properties, both in terms of separation and recovery and in detection by mass spectrometry (short peptides are outside the spectrometer's detection window). Furthermore, the small size of short peptides reduces the reliability of their identification by sequence homology. A systematic, non-targeted method has been developed for identifying a large number of short peptides in complex mixtures such as food matrices and protein hydrolysates.

[0243] Our developed method is based on two aspects: implementing an analytical method that includes processing the sample to label short peptides via dansylation; and performing instrumental analysis by LC-MS / MS, followed by data processing to identify the sequence of the dansylated short peptides in the sample based on the dataset, thereby generating its... N Short peptides terminally tansunylated. The tansunylation step is preferably performed before the alkylation step because, without this step, it has been observed that amino acids containing readily oxidizable free thiol groups, particularly cysteine, are difficult to detect. This alkylation step serves to protect the free thiol groups and facilitate their detection after labeling. The data processing steps rely on using an adjusted and calculated catalogue of short peptides, which does not correspond to a catalogue containing peptides derived from the digestion of endogenous or unlabeled proteins (available directly online or via a computer database of proteins). in silico (Produced by digestion), as in the usual case; but corresponding to the production of its components. N A catalogue of all possible combinations of natural and modified amino acids of terminally tansylated dipeptides, tripeptides, and tetrapeptides, i.e., a catalogue of approximately 6,250,000 predetermined peptides for N = 50.

[0244] Materials and methods Sample processing The peptides (typically yeast protein hydrolysates) in a given sample are alkylated and then dansylated.

[0245] Alkylation The peptide is alkylated to protect peptides containing free thiol groups.

[0246] The plan is as follows: The sample was incubated with 100 mM ammonium bicarbonate (ABC) (pH 8.8) containing 5 mM iodoacetamide at room temperature and in the dark for 1 hour.

[0247] Dansyl chloride was used N End marker The plan is as follows: Suspend 1 mg of sample in 1 mL of 100 mM ammonium bicarbonate (ABC) buffer (pH 8.8).

[0248] Suspend 1 mg of sample in 1 mL of 100 mM ammonium bicarbonate (ABC) buffer (pH 8.8).

[0249] Take 25 µL of the suspended sample and mix the aliquot with 12.5 µL of acetonitrile (ACN) and 12.5 µL of 100 mM ABC buffer (pH 8.8).

[0250] Vortex the mixture.

[0251] Add 25 µL of a 18 mg / mL solution of dansyl chloride in ACN and vortex to homogenize.

[0252] Incubate at 40°C for 1 hour.

[0253] Excess dansyl chloride was removed by adding 5 µL of 250 mM NaOH. Stop reaction (Quenching step), and vortex to homogenize.

[0254] Heat the mixture at 40°C for 10 minutes.

[0255] Add 25 µL of 425 mM formic acid (FA) (diluted in 50 / 50 ACN / water solution) and vortex to homogenize.

[0256] Evaporation was performed using SpeedVac.

[0257] The dried product was then reduced in 100 µL of water.

[0258] Aggregates were removed by filtering the sample using a 96-well plate equipped with a PVDF membrane (0.45 µm pore size) (after wetting the pores with 100 µL of 70% EtOH and washing the pores twice with 200 µL of Milli-Q water). No drying was performed.

[0259] Instrumental analysis was performed by LC-MS / MS. The filtrate was diluted 1:10 in water + 0.1% FA, and 1 µL of the diluted solution was injected into the instrument.

[0260] Instrumental analysis using LC-MS / MS Dansylated peptides were analyzed by liquid chromatography coupled with tandem mass spectrometry. The steps of this analysis were as follows: a. Separation of dansyl-substituted peptides by liquid chromatography b. Detection of dansyl-terminated peptide ions (parent ions) by MS (mother ion spectroscopy) c. Fragmentation in MS / MS (spectral analysis of fragment ions derived from the parent ion).

[0261] Liquid chromatography The liquid chromatography method used was nano-LC using a C18 column (Acclaim™ PepMap™ 100 C18), a type commonly used in proteomics.

[0262] The gradient is maintained for more than one hour, such as two hours, to improve chromatographic resolution.

[0263] Detection Detection was performed using tandem mass spectrometry. The precursor ion was detected in MS and fragmented in MS / MS. A high-resolution orbital trap mass analyzer operating in positive ion mode was used.

[0264] The detector allows for the acquisition of a set of information for each peptide being tested, as described above.

[0265] Processing data The previously described methods for non-targeted identification of peptide sequences can, for example, be used by... Figure 4 The apparatuses shown are implemented using the information set obtained above.

[0266] result Characterize the short peptide protein content of the sample. Identify the sequences of all short peptides present in the sample (350 to 500 short peptides with different sequences). Performing the method allows for the characterization of short peptides in complex mixtures, as well as the role of said short peptides in the technical and sensory properties of the hydrolysate or food matrix in which they are present.

[0267] Example 2: Studies of short peptides in soybean and casein hydrolysates and in yeast extracts.

[0268] 1. Research Objectives In this example, the method of the present invention is used to characterize short peptides present in soybean and casein hydrolysates and yeast extracts from different manufacturers, the short peptides being intended for different applications and being derived from different sources according to various hydrolysis processes.

[0269] 2. Materials and Methods 2.1. Chemical products, reactants, and samples Ammonium bicarbonate (A6141), ACN (acetonitrile) (1000292500), glycine (G7403), FA (formic acid) (1.00264), 37% HCl (hydrochloric acid) (320331), iodoacetamide (I1149), NQS (sodium 1,2-naphthoquinone-4-sulfonate) (70382), NaOH (sodium hydroxide) (221465), and TFA (trifluoroacetic acid) (T6508) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DNS-Cl (dansyl chloride) (D0656) was supplied by TCI Chemicals Europe (Zwijndrecht, Belgium). Ultrapure water was prepared using a Milli-Q ZRQSVP3WW purification system from Merck Millipore (Burlington, MA, USA). AccQ-Tag derivatization kit (186003836), amino acid standard (WAT088122), eluent A (186003839), and eluent B (186003838) for determining total and free amino acids were obtained from Waters Corporation™ (Milford, MA, USA).

[0270] Various commercially available hydrolyzed samples were selected based on the initial raw materials (plant, animal, or microbial) and the hydrolysis process (fermentation, enzymatic hydrolysis, or autolysis), and these samples are presented in Table 2:

[0271] [Table 2]: Hydrolysis products included in this study a Kikkoman Co., Ltd.: Noda-Shi, Japan; b Suzi Wan Company: Mars Company, McLean, VA, USA; c Sigma-Aldrich, Inc.: Saint-Louis, MO, USA; d ITW Reagents: Monza, Italy;e Gibbs Corporation: Life Technologies Miami, FL, USA.

[0272] 2.2. Analysis by size exclusion chromatography To characterize the molecular size profiles of proteins and peptides in the hydrolysate, analysis was performed by size exclusion chromatography (SEC) at 25 °C using a Prominence-iLC-2030 compact HPLC system (228-65800-58, Shimadzu™, Kyoto, Japan) equipped with a Shodex Protein KW-802.5 silica column (563781, 8 mm × 300 mm, Phenomenex™, Torrance, CA, USA) coupled with UV detectors (210 nm and 280 nm). A 25 μL volume of the sample, prepared as a 1% (w / w) solution and filtered through a 0.45 μm cellulose acetate syringe filter, was injected into the column. Elution was performed at a maximum pressure of 5 MPa and 25 °C using a mobile phase containing 20% ​​aqueous ACN and 0.1% TFA at a flow rate of 0.5 mL / min. Various standards of different molecular weights, typically used in size exclusion chromatography at a concentration of 2 mg / mL, were analyzed to determine the molecular weight range and corresponding retention volumes. Data were processed using LabSolutions v5.106 software, and calibration curves were generated using GPC run-through software (Shimadzu™, Kyoto, Japan).

[0273] 2.3. Sample Preparation Procedure Liquid samples of Gibco™ Bacto™ yeast extract and naturally fermented soy sauce were lyophilized using an Alpha 2-4 LCS plus instrument (CHRIST, Osterode am Harz, Germany). 1 mg of each sample was weighed and dissolved in 100 μL of 50 mM iodoacetamide in ammonium bicarbonate (100 mM, pH 8.8) to alkylate the free thiol groups of cysteine. This reaction was maintained at room temperature for one hour and terminated by dilution with 900 μL of ammonium bicarbonate solution (100 mM, pH 8.8). 25 μL of the mixture was transferred to a new 1.5 mL Eppendorf tube and diluted with 12.5 µL of ACN and 12.5 µL of ammonium bicarbonate solution (100 mM, pH 8.8). Next, 25 µL of DNS-Cl solution (18 mg / mL in ACN) was added to the mixture and incubated at 40 °C for 1 hour. The derivatization reaction was quenched with 5 µL of NaOH solution (250 mM) and incubated at 40 °C for 10 minutes. Neutralization with NaOH was achieved by adding 25 µL of formic acid solution (425 mM, in ACN / H2O v / v). The mixture was dried using an Eppendorf™ ConcentratorPlus (Savant ISS110 concentrator; Hamburg, Germany). The labeled sample was then resuspended in 100 µL of 0.1% formic acid solution and filtered through a hydrophobic PVDF membrane filter plate (MultiScreen HTS® IP, 0.45 µm, MSIPS4510, Merck Millipore) using a PlatePrep 96-well vacuum filter station (Supelco™, Bellefonte, PA, USA) according to the manufacturer's instructions. The filtrate was diluted in 0.1% formic acid solution (1:10) for LC-MS / MS analysis.

[0274] 2.4. LC-MS / MS Analysis Method The labeled peptides were partially looped for 5 minutes with buffer A (5% ACN, 0.1% FA) at a flow rate of 10 µL / min into a pre-concentration trap (Thermo Scientific™, Acclaim PepMap 100 C18, 5 µm, 300 µm inner diameter × 5 mm). Separation was then performed by reversed-phase liquid chromatography using a nanoflow HPLC system (U3000 RSLC, Thermo Scientific™, Waltham, MA, USA) equipped with a C18 column (Acclaim PepMap 100 C18, 3 µm, 75 mm inner diameter × 500 mm). Separation was achieved using a 120-minute linear gradient of 5% to 50% buffer B (75% ACN and 0.1% FA) at a flow rate of 250 nL / min at 45 °C. The gradient termination includes a 10-minute wash with 99% buffer B, followed by reequilibration with buffer A. This system is coupled to a QExactive Plus mass spectrometer (Thermo Fisher Scientific™) via a nanoelectrospray ionization source for detection by tandem mass spectrometry. Resolution is 350–1500 at 70,000. m / z The mass range, an AGC target of 1e6, and a maximum ion injection time of 90 ms were used to apply the data-dependent acquisition (DDA) method. Ten MS / MS spectra were acquired per MS scan (TopN) with an AGC target of 5e5, a maximum ion injection time of 140 ms for MS / MS, and a resolution of 17,500. Other parameters, especially... m / z The separation window, dynamic drag exclusion, and normalized collision energy were each set to 2. m / z 30 seconds and 30, where the default charge state z is 1.

[0275] 2.5. Data Processing and Statistical Analysis The raw files corresponding to the analyzed samples are processed and analyzed using Short_pept, an internal parallel software tool written in Python. In short, this software uses Thermo Fisher Scientific's RawFile Reader (https: / / github.com / thermofisherlsms / RawFileReader) to extract data related to mass-to-charge ratio, retention time, signal intensity, and spectral number from the raw files. The software generates DNS-tagged sample libraries, which are then analyzed by the Short_pept software. The software generates short peptide libraries and fragment ion libraries with DNS tags containing chemical formulas and mass-to-charge ratios for identifying precursor ions, based on a predetermined library consisting of natural and modified amino acids (25 amino acids) and chemical elements.

[0276] Table 3 shows the dictionary of natural and modified amino acids used in the Short_pept software for calculating short peptide precursor mass and fragment ion libraries:

[0277] [Table 3] Table 4 defines the quality of elements in the Short_pept software used to calculate the composition of a library:

[0278] [Table 4] Table 5 shows the fragmentation functions of fragment ions used in the Short_pept software to calculate MS / MS spectra:

[0279] [Table 5] By analyzing the MS / MS spectra of fragmented precursors, de novo sequencing of fragmented precursor ions corresponding to theoretical libraries was performed (Seidler et al., 2010. De novo sequencing of peptides by MS / MS. Proteomics, 10(4), 634-649). After peak adjustment using the Savitsky-Golay filter, information on retention time, signal intensity, and number of spectra of annotated peptides was extracted from MS detections associated with the chromatograms (Savitzky et al., 1964. Smoothing and Differentiation of Data by Simplified Least Squares Procedures. Analytical Chemistry, 36(8), 1627-1639). For confidence annotation of sequences, a score based on the intensity of fragment ions in the MS / MS spectra was calculated. The maximum length of short peptides was set to 4 amino acids. The mass tolerance for peptide precursors (MS) and fragment ions (MS / MS) was set to 10 ppm. The study focuses on variable variations in lysine acetylation, methionine oxidation, and cysteine ​​carbamoyl methylation. Based on low... m / z Specific fragment ions in the region were manually distinguished between isoleucine and leucine (Jiang et al., 2020. A computational and experimental study of the fragmentation of l-leucine, l-isoleucine and l-allo-isoleucine under collision-induced dissociation tandem mass spectrometry. Analyst (Cambridge, UK), 145(20), 6632-6638). The experiment was performed in triplicate.

[0280] 3. Results and Discussion Protein hydrolysates are typically characterized by the size of the macromolecules, their peptide content (calculated based on total nitrogen and amino nitrogen), and their degree of hydrolysis. In the industry, calculating the amino nitrogen / total nitrogen ratio is standard practice for obtaining approximate values ​​for protein hydrolysis (Yi et al., 2021. Estimation of Degree of Hydrolysis of Protein Hydrolysates by Size Exclusion Chromatography. Food Analytical Methods, 14(4), 805-813). As shown in Table 2 below, naturally fermented soy sauces (SH-1 and SH-2) exhibit the highest degree of hydrolysis compared to other types of hydrolysates, reaching approximately 60%, regardless of the manufacturer. In this case, the high degree of hydrolysis is consistent with the results in the SEC spectra, where the low molecular weight fraction (<0.5 kDa) shows the highest enrichment, accounting for 60-65% of the molecular spectrum. However, the degree of hydrolysis of enzymatically digestible peptone (SH-3) derived from soybean is low, not exceeding 29%, which is related to the percentage (30.5%) of low molecular weight fractions (<0.5 kDa). For SH-3, the most prominent fractions in the SEC profile are in the 1–5 kDa range, which corresponds to oligopeptides.

[0281] The results are presented in Table 6.

[0282]

[0283] [Table 6]: Results of the comprehensive analysis applied to protein hydrolysates a The percentage indicated for the SEC fraction represents the relative peak area for each fraction detected using UV at 210 nm.

[0284] b The degree of hydrolysis is estimated by the ratio of amino nitrogen to total nitrogen.

[0285] For casein hydrolysates, the results clearly demonstrated a significant difference in hydrolysis between the two products (Table 6). SEC profiles showed that the low molecular weight fraction of the pancreatic digest of casein CH-2 (64%) was significantly greater than that of the enzymatically hydrolyzed casein CH-1 (35%). This result correlated with the degree of hydrolysis, with CH-2 showing a higher degree of hydrolysis (40.6%) than CH-1 (21.7%). This result could be explained by treatment differences associated with the use of different enzyme formulations, which produced two well-defined products with contrasting compositions. For yeast extracts (YE-1 to YE-4), subtle differences in the degree of hydrolysis were observed between the products (see Table 6), with a degree of hydrolysis of approximately 40%, regardless of the manufacturer. However, the molecular weight (MW) distributions shown in the SEC spectra are quite different: although the low molecular weight fraction is dominant in all samples, this fraction is more pronounced in samples YE-1 (64.3%) and YE-3 (57.5%) than in YE-2 (49.5%) and YE-4 (49.8%), suggesting that these products originate from different processes.

[0286] While providing an overview of the compositional differences, most notably represented by small peptides, these top-level analyses cannot be directly correlated with the functional properties of the products or their applications. In-depth molecular characterization allows for a more detailed depiction of these differences and is directly related to activity.

[0287] The protein hydrolysates studied contained high levels of small peptides (see Table 6), which could be analyzed using the methods of this invention.

[0288] As detailed above, the method relies on the non-targeted identification of short peptide sequences and particularly allows for their relative quantification. In this study, molecules were amine-labeled with DNS-Cl, followed by nLC-MS / MS analysis using high-resolution mass spectrometry. Results for each of the samples SH-1, SH-2, SH-3, CH-1, CH-2, YE-1, YE-2, YE-3, and YE-4, obtained in CSV format, include the identified short peptide sequence, its corresponding retention time, and its signal intensity (not shown here).

[0289] The results are presented in Figure 5 and Figure 6 And in Table 7 below:

[0290] [Table 7]: Distribution of short peptide content based on signal intensity in various protein hydrolysates. Values ​​without parentheses represent the mean relative signal intensity (%) of three technical replicates for each analyzed sample, while values ​​with parentheses correspond to the standard deviation of the relative signal intensity (%) of three technical replicates for each analyzed sample. Soybean hydrolysates SH-1, SH-2, and SH-3.

[0291] The method described above allows for the identification of 196, 152, and 216 unique short peptide sequences (i.e., dipeptides, tripeptides, and tetrapeptides) in soybean hydrolysates SH-1, SH-2, and SH-3, respectively. Figure 5 Comparison of the identified sequences revealed considerable overlap, with SH-1 and SH-2, naturally brewed soybean hydrolysates intended for culinary applications, sharing a significant amount of sequence information. Figure 6 A). These two products share fewer sequences with soybean hydrolysate SH-3, which is obtained through enzymatic hydrolysis and marketed for biotechnology applications. Most of the identified sequences correspond to dipeptides (90-92% signal intensity), followed by tripeptides (9-10% signal intensity) (Table 7). Tetrapeptides were detected, but these constitute a minor component of the soybean hydrolysate (0.1-0.2% signal intensity) (Table 7). Compared to fermented soybean hydrolysate, the enzymatic hydrolysate contains more tripeptides and fewer dipeptides. These findings are consistent with results regarding the degree of hydrolysis, which are 59.7%, 60.3%, and 28.8% for SH-1, SH-2, and SH-3, respectively.

[0292] Casein hydrolysates CH-1 and CH-2 For casein hydrolysates, 309 and 310 unique short peptide sequences (i.e., dipeptides, tripeptides, and tetrapeptides) were identified for CH-1 and CH-2, respectively. Figure 5 Of these, 154 sequences are common to both products. Figure 6 B). However, the peptide distribution based on length was completely different between the two casein hydrolysates (Table 7). While the enzymatically derived casein hydrolysate produced a higher proportion of dipeptides (62% signal intensity), followed by tripeptides (30% signal intensity) and tetrapeptides (8% signal intensity), the pancreatic-derived casein hydrolysate showed a higher proportion of tripeptides (52% signal intensity), followed by dipeptides (38% signal intensity) and tetrapeptides (10% signal intensity). This suggests that peptones derived from enzymatic or pancreatic digestion of casein have different short peptide compositions and potentially indicate different functional properties.

[0293] Yeast extracts YE-1, YE-2, YE-3 and YE-4 For yeast extract, 350 to 450 unique short peptide sequences (i.e., dipeptides, tripeptides, and tetrapeptides) were identified, revealing that compositional differences may be related to supplier-specific processes. Figure 5 YE-1 is the extract that exhibits the greatest diversity in short peptide sequences. It also shares the fewest peptides with other yeast extracts, such as... Figure 6 As shown in C. All yeast extracts were mainly enriched in dipeptides (83-88% signal intensity), followed by tripeptides (12-16% signal intensity) and tetrapeptides (0.3-0.6% signal intensity) (Table 7).

[0294] To the best of our knowledge, these results represent the most comprehensive characterization of short peptides that form complex mixtures in hydrolyzed matrices.

[0295] Based on these results, other tools can be used for further data processing and exploration, such as to study the theoretical physicochemical properties and theoretical biological or biochemical activities of the product under investigation (not described here).

[0296] In interpreting the specification and its related claims, terms such as “comprise,” “include,” “incorporate,” “contain,” “be,” and “have” should be interpreted in a non-exclusive manner.

[0297] The method is not limited to the exemplary embodiments described above by way of example only, but covers all variations that can be conceived by those skilled in the art within the scope of the following claims.

[0298] Although described through several detailed exemplary embodiments, the proposed methods and apparatus for implementing the methods include various variations, modifications, and improvements that will be apparent to those skilled in the art, and it should be understood that such variations, modifications, and improvements fall within the scope of this disclosure as defined by the following claims. Furthermore, the foregoing aspects and features may be implemented together, individually, or interchangeably, and all various combinations and sub-combinations of these aspects and features fall within the scope of this disclosure. Additionally, some of the systems and apparatuses described above may not incorporate all the modules and functions described with respect to preferred embodiments.

Claims

1. A method for non-targeted identification of peptide sequences comprising 2 to 6 amino acids present in a sample, said method being carried out by an apparatus comprising a processing circuit system and a memory, said memory comprising: - At least one predetermined peptide library, wherein the predetermined peptides comprise 2 to 6 amino acids, each predetermined peptide being defined by a peptide sequence, and for each predetermined peptide, the library comprising: The constituent amino acids of the peptide sequence and the total mass-to-charge ratio of the peptide sequence ( m / z The peptide sequence has at least one terminal amino acid, and the at least one terminal amino acid is labeled with a terminal tag. Fragment ions calculated based on the peptide sequence, each associated with a mass-to-charge ratio, and at least one or more of the calculated fragment ions containing the terminal label. - Information sets, each associated with a peptide (referred to as an analyte) present in the sample as measured by mass spectrometry, each analyte comprising a peptide sequence having at least one terminal amino acid with a terminal label, each information set of the analyte comprising at least one MS / MS spectrum, the at least one MS / MS spectrum comprising the total mass-to-charge ratio of the analyte and one or more mass-to-charge ratios of the analyte fragment ions. The test peptide and the predetermined peptide are labeled with the same end marker at the same end. For each information set of the peptide being tested, the method includes: / a / Based on the library and based on the at least one MS / MS spectrum, (110) the selection of predetermined peptides is determined, wherein the total mass-to-charge ratio of each predetermined peptide in the selection is equal to the total mass-to-charge ratio of the analyte peptide, and the error tolerance is less than a predetermined value. / b / The labeled terminal amino acid of the analyte peptide is determined (120) by comparing the mass-to-charge ratio of the analyte fragment ion with the calculated mass-to-charge ratio of the fragment ion of the selected predetermined peptide, provided that the error tolerance is below a predetermined value. / c / Based on the calculated fragment ions in the selection, the determined labeled terminal amino acids, and the fragment ions to be tested, at least one reconstructed peptide sequence of the peptide to be tested is determined (130). - Deliver (140) an identification list that identifies the test peptide in the sample, wherein each test peptide in the list is defined by a reconstructed peptide sequence.

2. The method according to the preceding claim, characterized in that, The calculated fragment ions are derived from the peptide sequence by applying a fragmentation method to the peptide sequence. The fragmentation method is preferably the same as the fragmentation method of the mass spectrometer.

3. The method according to any one of the preceding claims, characterized in that, The predetermined peptide library contains all possible peptide sequences consisting of 2 to 6 amino acids, which are obtained from a set of N amino acids; each sequence in the predetermined library defines a predetermined peptide of the library, and further includes a terminal amino acid with the terminal tag. The library preferably contains at least one predetermined peptide, which represents a plurality of charged predetermined peptides, each charged predetermined peptide having the same peptide sequence as the at least one predetermined peptide and a corresponding charge state.

4. The method according to any one of the preceding claims, characterized in that, The type of the terminal end of the labeled terminal amino acid is N End and / or C The end, preferably N End.

5. The method according to any one of the claims, characterized in that, The total mass-to-charge ratio of the predetermined peptide ( m / z The mass-to-charge ratio is determined based on the constitutive amino acid residues in the sequence of the predetermined peptide.

6. The method according to any one of the preceding claims, characterized in that, The mass spectrometer is a mass spectrometer (MS / MS) with tandem analysis capability, preferably a mass spectrometer coupled with liquid chromatography (LC).

7. The method according to any one of the preceding claims, characterized in that, In / c / , at least one peptide sequence of the peptide being tested is reconstructed by the following: - The identified terminal amino acid of the label, - A comparison between the mass-to-charge ratio of the fragment ion being measured and the calculated mass-to-charge ratio of the fragment ion.

8. The method according to any one of the preceding claims, characterized in that, The memory includes at least a first library and a second library, the first library containing the constitutive amino acids of the peptide sequence and the total mass-to-charge ratio of the peptide sequence. m / z The peptide sequence has at least one terminal amino acid, and the at least one terminal amino acid is marked with the terminal tag; The second library is determined only for the selected predetermined peptides, the second library containing at least the selected predetermined peptides and containing fragment ions calculated based on the peptide sequences of the selected predetermined peptides, the calculated fragment ions being associated with mass-to-charge ratios, and at least one or more of the calculated fragment ions containing the terminal markers.

9. The method according to the preceding claim in conjunction with claim 2 or claim 3, characterized in that, The second library was determined based on at least one fragmentation method applied to the predetermined peptide in the selection.

10. The method according to any one of the preceding claims, characterized in that, When multiple reconstructed peptide sequences are determined for the same test peptide in / c / , the method further includes: / d / A ​​score is calculated for each reconstructed peptide sequence based on the information set associated with the peptide being tested and based on the reconstructed peptide sequence. The reconstructed peptide sequence of the test peptide was selected based on a comparison of the scores of the reconstructed peptide sequences, and the peptide sequence with the highest score was selected.

11. The method according to the preceding claim, characterized in that, Each mass-to-charge ratio of the analyte fragment ion is correlated with its intensity in the MS / MS spectrum, and the score for each reconstructed peptide sequence of the same analyte peptide is determined based on the following equation: Preferably, the peptide sequence score is calculated using only the tested fragment ions with the terminal label.

12. The method according to any one of the preceding claims, characterized in that, In / b / , one or more labeled terminal amino acids are identified, and in / c / , for each identified labeled terminal amino acid, at least one reconstructed peptide sequence of the test peptide is determined based on the calculated fragment ion in the selection, the identified labeled terminal amino acid, and the test fragment ion.

13. The method according to any one of the preceding claims, characterized in that, The list contains retention times associated with the test peptide, each retention time being obtained from the information set of the reconstructed peptide sequence used to determine the test peptide.

14. The method according to any one of the preceding claims, characterized in that, Each peptide in the list is associated with the information set of the reconstructed peptide sequence used to identify the peptide by means of identification methods.

15. The method according to any one of the preceding claims, characterized in that, The list contains intensities associated with each of the test peptides, each intensity obtained from the information set of reconstructed peptide sequences used to determine the test peptide, and each test peptide in the list contains a proportion value indicating the proportion of the peptide in the mixture, the proportion value "Qp" of each test peptide in the list being calculated based on the following: 。 16. The method according to any one of the preceding claims, characterized in that, The peptide is a natural peptide and / or a synthetic peptide.

17. The method according to any one of the preceding claims, characterized in that, The peptide contains at least one labeled terminal amino acid and one or more amino acids selected from protein-derived amino acids or modified amino acids.

18. The method according to any one of the preceding claims, characterized in that, The sample is a protein hydrolysate, preferably a yeast protein hydrolysate, a plant protein hydrolysate, or an animal protein hydrolysate.

19. The method according to any one of the preceding claims, characterized in that, The terminal amino acid of the label is marked with N End-marked N terminal amino acids, the N The terminal label is selected from dansyl, dabsyl, 1-naphthyl isocyanate (IsoC), or 1-naphthyl isothiocyanate, each of which is optionally marked with carbon-13 ( 13 C) Deuterium (D) Nitrogen-15 ( 15 N) and / or sulfur-34 ( 34 One or more atomic tags in S).

20. The method according to any one of the preceding claims, characterized in that, The terminal amino acid of the label is marked with C End-marked C terminal amino acids, the C End markings are selected from 2-( N,N' -dimethylamino)ethyl-1-amine (DMED) and 3-( N,N' -dimethylamino)prop-1-amine (DMAPA), each of these terminal tags is optionally represented by carbon-13 ( 13 C), deuterium (D) and / or nitrogen-15 ( 15 One or more atomic labels in N).

21. The method according to any one of the preceding claims, characterized in that, The peptide has undergone treatment to protect free thiol groups, typically alkylation, and the mass-to-charge ratio of the predetermined peptide in the library takes into account the treatment to protect the free thiol groups of cysteine.

22. A method for analyzing peptides having 2 to 6 amino acids in a sample, the method comprising: i) A sample processing step, the sample processing step comprising at least one step i1) of labeling the ends of the peptides in the sample by contacting the sample with an end label. ii) Analytical steps, wherein the analytical steps utilize liquid chromatography coupled with a mass spectrometer, preferably a mass spectrometer (MS / MS) with tandem analytical capabilities. iii) A data processing step, wherein the data processing step implements the method for non-targeted identification of peptide sequences according to any one of the preceding claims.

23. The method according to the preceding claim, wherein the sample processing step i) further comprises step i2) of alkylating the peptide by contacting the peptide of the sample with an alkylating agent, and wherein step i2) is preferably prior to step i1).

24. The method according to any one of claims 22 or 23, characterized in that, The liquid chromatography method is reversed-phase liquid chromatography, preferably performed using a gradient of at least one hour.

25. The method according to any one of claims 23 to 24, characterized in that, The mass spectrometer is equipped with a mass analyzer, which is preferably selected from a quadrupole ion trap mass analyzer and a triple quadrupole mass analyzer; and more preferably, the mass spectrometer is equipped with a high-resolution mass analyzer, which is selected from an orbital trap mass analyzer, a Q-TOF mass analyzer, a TIMS-TOF mass analyzer, and an FT-ICR mass analyzer.

26. A computer program comprising instructions for implementing the method according to any one of claims 1 to 21 when the program is executed by a processor.

27. A non-transitory computer-readable storage medium storing a program for implementing the method according to any one of claims 1 to 21 when executed by a processor.

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