Novel method for identifying diagnostic marker peak of protein citrullination through mass spectrum

Abstract

The invention relates to a novel method for identifying a diagnostic marker peak of protein citrullination by mass spectrometry. A gas phase fragmentation pathway of citrullinated peptide is explored, a characteristic neutral loss is found, namely, the neutral loss of 43.0058 Da is matched with release of an isocyanate group of citrullinated ureido, the two modifications can be distinguished when the corresponding neutral loss is detected, and citrullinated modification can be accurately and quickly confirmed. When secondary mass spectrum data are retrieved, an anti-library is additionally added to calculate the false positive rate caused by random matching, and a common pollution library is added into a database to eliminate the influence of pollution protein in an identification result; the mass error tolerance is set in a targeted manner, and the accuracy of mass spectrum data retrieval and the diagnosis accuracy of protein citrullination are improved. The process of converting arginine into citrulline can be diagnosed with high specificity, the technology is a key technology for realizing polypeptide sequencing and post-translational modification analysis, and the specific citrulline part in the protein can be determined.

Description

Technical Field

[0001] The present invention relates to the field of mass spectrometry detection, and in particular to a new method for mass spectrometry identification of diagnostic marker peaks of protein citrullination. Background Art

[0002] Citrullination is a post-translational modification in mammalian cells, i.e., a chemical modification of proteins after translation, through which peptidylarginine residues are converted to citrulline (Cit), catalyzed by the peptidylarginine deiminase (PAD) family. This modification eliminates the positive charge on arginine (a process known as deimination), eliminating intra- and intermolecular electrostatic interactions that can regulate protein structure and function. Arginine citrullination is shown in formula (1):

[0003]

[0004] Citrullination is involved in diverse cellular processes, including gene regulation, apoptosis, and skin homeostasis. Altered citrullination levels have been implicated in the pathogenesis of diseases such as multiple sclerosis, rheumatoid arthritis (RA), psoriasis, and Alzheimer's disease. In RA, elevated protein citrullination in synovial joints has been shown to trigger an autoimmune response and lead to joint damage. Anti-citrullinated peptide IgG detection is highly specific for RA in patients and is widely used in the clinical diagnosis of RA.

[0005] Although new biological studies on citrullination are emerging all the time, the physiological role of PAD enzymes and protein citrullination is far less well understood than other protein post-translational modifications. Of the 511,876 results found under the PTM category of the UniProt knowledge database, only 219 (0.04%) were classified as citrullination. This is partly due to the lack of robust biochemical enrichment and detection methods. Commonly used methods for studying citrullination, such as immunodetection and visualization based on rhodamine-phenylacetaldehyde, are convenient ways to assess citrullination at the protein level, but these methods have serious limitations. Antibodies against specific citrullinated proteins (such as citrullinated histones or citrullinated fibrinogen) cannot recognize citrullination at the global protein level in vivo, which is necessary to study the hallmarks of RA. Peptidyl citrulline antibodies (such as F95 monoclonal antibody), denatured citrulline antibodies (Senshu method), and rhodamine-phenylacetaldehyde can broadly detect citrullinated proteins but cannot provide information about the identity of citrullinated proteins. Despite the obvious biological significance of protein citrullination, less than ten proteins have been identified as citrullinated in vivo, including histones (H3, H2A, H4), nuclear phospholipids, myelin, and keratin. Although anti-citrulline antibodies can sensitively detect citrullination of proteins, the definition of the exact site of modification can only be established by mass spectrometry methods.

[0006] During citrullination, all fragment ions containing citrulline residues have a mass increase of 0.9840 Da compared to the unmodified arginine-containing peptide. These ΔM values ​​are very useful for the annotation of MS / MS spectra of post-translationally modified peptides, as they aid in the identification and assignment of the modified residues. However, although these features indicate that citrullinated proteins can be directly characterized by MS / MS, the mass increase of 0.9840 Da is not specific for citrullination. This exact mass increase also occurs for one of the most common modifications, the deamidation of asparagine (Asn) or glutamine (Gln), which is a spontaneous, non-enzymatic conversion of uncharged side chain residues. The presence of deamidated and citrullinated residues in the same peptide can lead to interferences during the separation of precursor ions and can result in incorrect assignment of the modified amino acid, leading to mixed or contaminated MS / MS spectra. In fact, deamidation of Asn / Gln occurs more frequently than citrullination. Citrullination can also be mistaken for 13 C isotope, because 13 The mass increment of the C isotope is 1.0036 Da.

[0007] The above factors make it more complicated to specifically distinguish citrullination and deamidation by mass spectrometry, so there is an urgent need for an accurate and rapid method to distinguish citrullination from other interfering modifications. Summary of the invention

[0008] To overcome the above defects of the prior art, the present invention provides a new method for mass spectrometry identification of diagnostic marker peaks of protein citrullination. The present invention explores the gas phase fragmentation pathway of citrullinated peptides by electrospray ionization-tandem mass spectrometry and finds a characteristic neutral loss, i.e., a neutral loss of 43.0058 Da matches the release of the isocyanate (HN=CO) group of the citrulline urea group. The neutral loss of HNCO can provide a specific marker for identifying citrullination.

[0009] Neutral loss is a special comparison test, because mass spectrometry detects ions, that is, only those that exist in ionic state can be detected in mass spectrometry. Some compounds will lose NH3, H2O or other uncharged groups with a little energy, and these lost groups are naturally invisible (because they are not ions). Therefore, for this type of substance, the parent ion and ion will differ by one NH3 or H2O or other uncharged group, and this loss is very common, and the signals of the parent and daughter ions can exist stably.

[0010] Liquid chromatography mass spectrometry (LC-MS / MS) can analyze a mixture of thousands of peptides, obtain the mass-to-charge ratio (m / z) of each peptide precursor ion (MS1), and fragment the most abundant precursor for MS / MS (secondary spectrum) detection. The resulting fragment ions are recorded as MS / MS spectra, which are searched and matched against theoretical databases. During this search, the masses of the observed precursor and fragment product ions are compared with the masses of the theoretical protein digest, and the amino acid sequence and statistical confidence of the sequence match are returned. This accuracy makes MS / MS a key technology for peptide sequencing and post-translational modification analysis because it facilitates the mass determination and sequencing of peptides, thereby enabling the detection of post-translational modifications at specific sites. Most post-translational modifications can be detected by changes in the molecular weight of the modified amino acid relative to the unmodified form, and these increases or deficiencies in mass can be detected by MS (mass spectrometry). In fact, MS is currently the gold standard for confirming citrullination and is also the standard for determining the identity and site of citrullination in proteins. However, there are still some important factors to consider to avoid misleading interpretations of mass spectrometry data.

[0011] The mass change of peptide arginine converted to citrulline is small (+0.9840Da), and is the same as the mass change of deamidation (+0.9840Da) of asparagine (N, Asn) and glutamine (Q, Gln) residues, which makes mass spectrometry detection challenging, especially on low-resolution mass spectrometers. Therefore, the present invention changes the idea and studies the gas phase fragmentation pathway of citrullinated peptides, and finds that there is a neutral loss process in this pathway, as shown in formula (2):

[0012]

[0013] During this neutral loss, the isocyanate (HN=CO) group of the citrulline urea group is released, resulting in a characteristic neutral loss of 43.0058 Da. The neutral loss of HNCO can provide a specific marker for identifying citrullination.

[0014] The present invention conducts experimental research on the fragmentation energy entering the HCD collision cell and finds that when the fragmentation energy is 45%, the maximum 43.0058Da neutral loss can be detected; under this condition, the accuracy of identifying the arginine citrullination of proteins is the highest.

[0015] Citrullination can also be mistaken for 13 C isotope, because 13 The mass increment of the C isotope is 1.0036 Da. 13 The mass difference of C isotopes is 0.0196Da. The parent ion is generally within 30 amino acids, and the average molecular weight of amino acids is 128Da. If we want to separate citrullinated and 13 To distinguish C isotopes, it is necessary to distinguish at least 0.0196 / (30*128)=5ppm. 13 Misidentification of C isotopes can be circumvented by searching the database with a sufficiently small precursor mass tolerance, such as <5ppm. There are usually two sets of mass tolerances input into the database search: peptide mass tolerance and fragment mass tolerance. The peptide mass tolerance setting is based on the mass accuracy of the analyzer used to measure the mass of the precursor ion in the MS scan. The fragment mass error is based on the mass accuracy of the analyzer used to measure the fragment ions in the MS / MS scan. This parameter may be referred to as the MS / MS error, fragment mass error, or product ion error. The ideal precursor mass tolerance for citrullination would be <5ppm, with an MS / MS tolerance of 0.6Da.

[0016] The present invention provides a method for identifying protein citrullination by mass spectrometry, which identifies the characteristic neutral loss of gas phase fragmentation of citrullinated peptides in mass spectrometry. The neutral loss is the release of the isocyanate group of citrulline urea group, which has a size of 43.0058 Da.

[0017] Furthermore, the above-mentioned method for identifying protein citrullination by mass spectrometry comprises the steps of:

[0018] (1) proteolysis of proteins into peptides and preparation of peptide solutions;

[0019] (2) The peptide solution was detected using LC-MS / MS;

[0020] (3) Mass spectrometry data were searched using software;

[0021] (4) Extracting the secondary spectrum to confirm the signature peak of the neutral loss.

[0022] Furthermore, the peptide solution in step (1) is prepared by taking 1 mg of the peptide and dissolving it in 0.1% formic acid aqueous solution to a solution of 2.5 ng / μl.

[0023] Furthermore, in step (2), the mass spectrometer ion source is an NSI ion source; the scanning resolution is set to 120,000; and the secondary scanning resolution is set to 15,000.

[0024] Furthermore, in step (2), after the primary mass spectrometry scan, the top 10 peptide precursor ions with the highest signal intensity are selected and sequentially enter the HCD collision cell for fragmentation using 28% fragmentation energy, and secondary mass spectrometry analysis is also performed sequentially.

[0025] Furthermore, the mass spectrometry parameters in step (2) are: automatic gain control is set to 5E4, signal threshold is set to 2.5E5 ions / s, maximum injection time is set to 40 ms, and dynamic exclusion time of tandem mass spectrometry scanning is set to 30 s.

[0026] Furthermore, in the search parameters in step (3), the database additionally adds a counter-library and a contamination library; the mass error tolerance of the primary parent ion input into the First search and Main search in the database search is set to 5ppm and 4.5ppm respectively, and the mass error tolerance of the secondary fragment ion is 5ppm.

[0027] Furthermore, in the search parameters in step (3), the enzyme cleavage method is set to Trypsin / P; the number of missed cleavage sites is set to 2; the minimum length of the peptide is set to 7 amino acid residues; and the maximum number of peptide modifications is set to 5.

[0028] Furthermore, in the search parameters in step (3), cysteine ​​alkylation is set as a fixed modification, and the variable modifications are one or more of methionine oxidation, protein N-terminal acetylation, deamidation, and lysine citrullination.

[0029] Furthermore, in the search parameters in step (3), the false positive rates of protein identification and peptide matching spectrum identification were both set to 1%.

[0030] The beneficial effects of the present invention are:

[0031] 1. In the present invention, we explored the gas phase fragmentation pathway of citrullinated peptides by electrospray ionization-tandem mass spectrometry and found a characteristic neutral loss, that is, the neutral loss of 43.0058 Da matched the release of the isocyanate (HN=CO) group of the citrulline urea group. That is, the detection of the corresponding neutral loss can distinguish the two modifications, and the citrullination modification can be accurately and quickly confirmed, eliminating the interference of aspartic acid deamidation modification.

[0032] 2. When searching the secondary mass spectrometry data, an additional anti-library was added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library was added to the database to eliminate the influence of contaminating proteins in the identification results; the mass error tolerance of the primary parent ion and the secondary fragment was set specifically to improve the accuracy of mass spectrometry data retrieval and the diagnostic accuracy of protein citrullination, eliminating 13 C isotope interference.

[0033] 3. The process of arginine conversion to citrulline can be diagnosed with high specificity. This technology is a key technology for peptide sequencing and post-translational modification analysis, and can determine the specific site of citrullination in proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 :Peptide AVCMLSNTTAIAEAWAR secondary mass spectrum

[0035] Figure 2 :Peptide AVCMLSN[Dea]TTAIAEAWAR secondary mass spectrum

[0036] Figure 3 :Peptide AVCMLSNTTAIAEAWAR[Cit] secondary mass spectrum DETAILED DESCRIPTION

[0037] The following is a further explanation of the method for identifying protein citrullination by mass spectrometry of the present invention in conjunction with specific examples and the accompanying drawings, but the contents of the following examples should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents used in the embodiments of the present invention are all commercially available.

[0038] Example 1: Mass spectrometry identification of citrullination modification

[0039] 1. Processing of standard peptides:

[0040] Take 1 mg each of the synthesized 95% pure standard peptides AVCMLSNTTAIAEAWAR (without any modification), AVCMLSN[Dea]TTAIAEAWAR (with asparagine deamidation), and AVCMLSNTTAIAEAWAR[Cit] (with arginine citrullination), and dissolve them in 0.1% formic acid solution to a solution of 2.5 ng / μl;

[0041]

[0042] Formula (3) Asparagine deamidation

[0043] 2. LC-MS detection:

[0044] The peptide solution was separated using an EASY-nLC 1200 ultra-high performance liquid phase system.

[0045] Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile.

[0046] Liquid phase gradient settings: 0-12min, 4% to 23% B; 12-22min, 23% to 35% B; 22-26min, 35% to 80% B; 26-30min, 80% B, flow rate maintained at 500nL / min. The length of the homemade chromatographic column is 25cm, the inner diameter is 100μm, and the filler specification is 1.9μm / ReproSil-PurC18, column temperature was set at 50°C.

[0047] The peptides were separated by ultra-high performance liquid chromatography (UPLC) and injected into the NSI ion source for ionization. TM HF-X mass spectrometry was used for analysis. The ion source voltage was set to 2.1 kV, in positive ion mode, and the peptide parent ion and its secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set to 400-1500 m / z, the scan resolution was set to 120,000, and the secondary scan resolution was set to 15,000.

[0048] The data acquisition mode used a data-dependent scanning (DDA) program, that is, after the primary scan, the top 10 peptide precursor ions with the highest signal intensity were selected and sequentially entered the HCD collision cell for fragmentation using 45% fragmentation energy, and secondary mass spectrometry analysis was also performed in sequence.

[0049] In order to improve the effective utilization of mass spectrometry, the secondary mass spectrometry conditions were as follows: automatic gain control (AGC) was set to 5E4, signal threshold was set to 2.5E5 ions / s, maximum injection time was set to 40 ms, and dynamic exclusion time of tandem mass spectrometry scanning was set to 30 s to avoid repeated scanning of parent ions.

[0050] 3. Search the database:

[0051] The secondary mass spectrometry data were retrieved using Maxquant (v1.6.15.0).

[0052] Search parameter settings: the database is Homo_sapiens_9606_SP_20230103.fasta (20389 sequences), a reverse library is added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library is added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme cutting mode is set to Trypsin / P; the number of missed cutting sites is set to 2; the minimum length of the peptide is set to 7 amino acid residues; the maximum number of peptide modifications is set to 5; the mass error tolerance of the primary parent ion of First search and Mainsearch is set to 5ppm and 4.5ppm respectively, and the mass error tolerance of the secondary fragment ion is 5ppm. Cysteine ​​alkylation Carbamidomethyl (C) is set as a fixed modification, and the variable modifications are oxidation of methionine, acetylation of the protein N-terminus, deamidation (NQ), and lysine citrullination. The FDR of protein identification and PSM identification is set to 1%.

[0053] 4. Confirmation of landmark peaks:

[0054] The following is a detailed description of the secondary spectrum data of the three peptides:

[0055] 1. The secondary spectrum of peptide AVCMLSNTTAIAEAWAR is as follows Figure 1 As shown, it does not contain any modification, and the b and y ion fragments are rich in information, but there is no corresponding -43.0058Da neutral loss ion.

[0056] 2. The secondary spectrum of AVCMLSN[Dea]TTAIAEAWAR is as follows Figure 2 As shown, asparagine deamidated peptides, including y12, y13, and y14 with N deamidation, can be identified, but there is no corresponding neutral loss ion of -43.0058 Da.

[0057] 3. The secondary spectrum of AVCMLSNTTAIAEAWAR[Cit] is as follows Figure 3 As shown, the arginine citrullinated peptides, including R-citrullinated y11, y12, and y13, can be identified, and have corresponding neutral loss ions of -43.0058Da (HN=CO). After the neutral loss of y11, y12, and y13, they are y11*, y12*, and y13*, respectively, and their mass differences are all -43.0058Da.

[0058] V. Conclusion:

[0059] according to Figure 3 The method provided by the present invention is to identify the neutral loss ion of -43.0058Da (HN=CO) in the secondary spectrum to determine whether R citrullination exists.

[0060] contrast Figure 1 and Figure 3 , it can be found that: containing arginine citrullinated peptides, the mass spectrum peak containing R citrullinated group ions can be detected, for example Figure 3 The y11* in the figure is obtained by losing 43.0058Da of the y11 peak, which proves that the method provided by the present invention can realize the diagnosis of protein citrullination.

[0061] contrast Figure 2 and Figure 3 , we can find: AVCMLSN[Dea]TTAIAEAWAR and

[0062] Although AVCMLSNTTAIAEAWAR[Cit] both have a mass change of +0.9840Da (N deamidation or R citrullination), the present method can distinguish the two through the neutral loss of -43.0058Da. Only the arginine citrullinated peptide segment has the above neutral loss. The method provided by the present invention can realize the diagnosis of protein citrullination with high discrimination, eliminating the possibility of misdiagnosis.

[0063] Example 2: Mass spectrometry conditions for detecting neutral loss

[0064] Neutral loss originates from the first-stage scan and then enters the collision cell for fragmentation, and then is detected by the second-stage mass spectrometer. In the present invention, we optimized the mass spectrometry detection parameters of the arginine citrullinated peptide segment. In the HCD fragmentation mode, different fragmentation energies were set respectively. The other methods were the same as those described in Example 1. The AVCMLSNTTAIAEAWAR[Cit] peptide segment solution obtained in Example 1 was detected, the mass spectrum was analyzed, and the mass spectrum intensity of the -43.0058Da (HN=CO) neutral loss ion was statistically analyzed. The experimental results are shown in Table 1.

[0065] Table 1: Fragmentation energy and mass spectrum intensity of -43.0058Da neutral loss ion

[0066]

[0067] The results showed that when the fragmentation energy was 45%, more citrullinated -43.0058Da (HN=CO) neutral loss ions could be detected.

[0068] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for identifying protein citrullination by mass spectrometry, characterized in that: A neutral loss characteristic of gas phase fragmentation of citrullinated peptides was identified in the mass spectrum as the release of the isocyanate group of the citrullinated urea group with a size of 43.0058 Da.

2. The method according to claim 1, characterized in that: Includes steps: (1) proteolysis of proteins into peptides and preparation of peptide solutions; (2) The peptide solution was detected using LC-MS / MS; (3) Mass spectrometry data were searched using software; (4) Extracting the secondary spectrum to confirm the signature peak of the neutral loss.

3. The method according to claim 2, characterized in that: The preparation method of the peptide solution in step (1) is as follows: 1 mg of the peptide is taken and dissolved into a 2.5 ng / μl solution using 0.1% formic acid aqueous solution.

4. The method according to claim 2, characterized in that: In step (2), the mass spectrometer ion source is an NSI ion source; the scanning resolution is set to 120,000; and the secondary scanning resolution is set to 15,000.

5. The method according to claim 4, characterized in that: In step (2), after the mass spectrometer is scanned at the primary level, the top 10 peptide precursor ions with the highest signal intensity are selected and sequentially enter the HCD collision cell for fragmentation using 45% fragmentation energy for secondary mass spectrometry analysis.

6. The method according to claim 5, characterized in that The mass spectrometry parameters in step (2) were: automatic gain control set to 5E4, signal threshold set to 2.5E5 ions / s, maximum injection time set to 40 ms, and dynamic exclusion time of tandem mass spectrometry scanning set to 30 s.

7. The method according to claim 2, characterized in that: In the search parameters in step (3), the database additionally adds the anti-library and the contamination library; the mass error tolerance of the primary parent ion input into the First search and Main search in the database search is set to 5ppm and 4.5ppm respectively, and the mass error tolerance of the secondary fragment ion is 5ppm.

8. The method according to claim 7, characterized in that: In the search parameters in step (3), the enzyme cleavage method was set to Trypsin / P; the number of missed cleavage sites was set to 2; the minimum length of the peptide was set to 7 amino acid residues; and the maximum number of peptide modifications was set to 5.

9. The method according to claim 8, characterized in that In the search parameters in step (3), cysteine ​​alkylation is set as a fixed modification, and the variable modifications are one or more of methionine oxidation, protein N-terminal acetylation, deamidation, and lysine citrullination.

10. The method according to claim 9, characterized in that In the search parameters in step (3), the false positive rates of protein identification and peptide matching spectrum identification were both set to 1%.

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