A heavy-labeled standard MHC I immune peptide mixture and its application in immune peptide detection

Abstract

The present invention proposes a re-labeled standard MHC I immune peptide mixture and its application in immune peptide detection, wherein the re-labeled standard MHC I immune peptide mixture comprises 10 immune peptides, the amino acid sequences of which are shown in SEQ ID NO.1-10; a non-terminal amino acid in each immune peptide is 13 C5 and 15 N label. The present invention uses non-terminal amino acids 13 C5 and 15 When the N-labeled heavy-labeled standard MHC I immune peptide mixture is used for immune peptide detection, it can be distinguished from non-heavy-labeled peptides in the mass spectrometry, and does not affect their retention properties in reversed-phase chromatography. It has the characteristics of good solubility, good liquid-mass response, wide chromatographic retention range, and wide application range. It can be widely used in the mass spectrometry method quality control process for immune peptide detection, the correction of information such as immune peptide retention time and quantification, and the development of immune peptide mass spectrometry methods and other practical work in immune peptide research.

Description

Technical Field

[0001] The invention relates to the technical field of immune peptide detection, in particular to a heavy-labeled standard MHC I immune peptide segment mixture and application thereof in immune peptide detection. Background Art

[0002] Immune peptides are peptides presented by cells through the major histocompatibility complex (MHC) on the cell surface. There are two main types of MHC molecules on the cell surface, namely MHC-I and MHC-II. There are significant differences in the structure of the peptide binding region between MHC I and MHC II molecules, resulting in different characteristics of immune peptides bound to MHC-I / II.

[0003] The peptide binding region of the MHC-I molecule is narrow, so the immune peptides it binds are usually 8-12 in length and are relatively short. Analysis has found that the peptide sequences bound by different MHC I molecules have clear positional regularity, the end of the sequence often lacks basic amino acids, and the abundance in the body is usually low; the peptide binding region of the MHC-II molecule is open, and the length of the peptide that can be bound is mainly 13-17, and the ionization effect in the mass spectrometry is better. The mass spectrometry detection method of immune peptides is to enrich the complex of MHC and peptides through antibodies, and then separate the MHC molecules and peptides, and then take the peptides for direct mass spectrometry identification. Therefore, the peptide sequence characteristics of immune peptides are closely related to HLA typing.

[0004] The sequence characteristics of MHCI immune peptides make them difficult to be ionized and fragmented to produce ideal secondary spectra, which affects subsequent data analysis. The abundance of MHCI immune peptides is low, which directly affects the mass spectrometry detection signal. These factors directly affect the quality of mass spectrometry detection spectra and become a bottleneck in the development of immune peptidomics (Escobar et al., 2011; Kote et al., 2020).

[0005] In the field of proteomics, the commonly used iRT kit (Biognosys) can be used for retention time and protein quantification calibration of proteomics detection. These standard peptides are non-weighted and end with lysine (K) or arginine (R), which are suitable for standard proteomics analysis processes, but not for applications such as quality control in immunopeptidome research.

[0006] Therefore, it is particularly important to develop a set of standard peptides and application methods suitable for the immune peptide group, which can realize the quality control of mass spectrometry methods and data analysis for immune peptide detection, and standard MHC I immune standard peptides that can be added to immune peptide samples for real-time monitoring without affecting the mass spectrometry detection depth of the immune peptide group of the actual sample. Summary of the invention

[0007] In view of the above, the present invention designs a mixed heavy-label standard peptide for MHCI binding immune peptide (hereinafter referred to as MHCI peptide) based on the prediction algorithm of immune peptide, which can be applied to the development and optimization of mass spectrometry methods and quality control of liquid-mass spectrometry methods in MHCI immune peptide research.

[0008] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides a re-labeled standard MHC I immune peptide mixture, including 10 immune peptides, the amino acid sequences of which are shown in SEQ ID NO.1-10; one non-terminal amino acid in each immune peptide is 13 C5 and 15 N mark.

[0009] On the basis of the above technical solution, preferably, the labeled amino acids are leucine, proline and valine.

[0010] On the basis of the above technical solution, preferably, the valine at position 6 in the amino acid sequence shown in SEQ ID NO.1 is 13 C5 and 15 N-labeled; the valine at position 3 in the amino acid sequence shown in SEQ ID NO.2 was 13 C5 and 15 N-labeled; the valine at position 8 in the amino acid sequence shown in SEQ ID NO.3 was 13 C5 and 15 N-labeled; proline at position 5 in the amino acid sequence shown in SEQ ID NO.4 was 13 C5 and 15 N-labeled; the valine at position 7 in the amino acid sequence shown in SEQ ID NO.5 was 13 C5 and 15 N-labeled; the valine at position 4 in the amino acid sequence shown in SEQ ID NO.6 was 13 C5 and 15 N labeling; the valine at position 3 in the amino acid sequence shown in SEQ ID NO.7 was 13 C5 and 15 N-labeled; Leucine at position 7 in the amino acid sequence shown in SEQ ID NO.8 was labeled 13 C5 and 15 N-labeled; proline at position 7 in the amino acid sequence shown in SEQ ID NO.9 was 13 C5 and 15 N-labeled; proline at position 7 in the amino acid sequence shown in SEQ ID NO.10 was 13 C5 and 15Marked with N. The molar ratio of the 10 immune peptides shown in SEQ ID NOs. 1-10 is 1:1:1:1:1:1:1:10:10:10.

[0011] In a second aspect, the present invention also provides an application of a mixed standard re-labeled MHC I immune peptide segment in HLA I immune peptide detection.

[0012] In a third aspect, the present invention further provides a HLA I immune peptide detection kit, comprising an HLA I immune peptide sample and the re-labeled standard MHC I immune peptide mixture according to any one of claims 1 to 4.

[0013] Based on the above technical solution, preferably, the dosage of the re-labeled standard MHC I immune peptide mixture is 0.8-8 fmol / μL.

[0014] The re-labeled standard MHC I immune peptide mixture of the present invention and its application in immune peptide detection have the following beneficial effects compared with the prior art:

[0015] The present invention uses non-terminal amino acids 13 C5 and 15 When the N-labeled heavy-labeled standard MHC I immune peptide mixture is used for immune peptide detection, it can be distinguished from non-heavy-labeled peptides in the mass spectrum and does not affect their retention properties in reversed-phase chromatography. It has the characteristics of good liquid-mass response and wide chromatographic retention range. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figures 1 to 10 The following are the secondary mass spectra (MSMS) of 10 heavy-labeled single-labeled peptides represented by SEQ ID NO. 1-10;

[0018] Fig.11 This is the retention and response diagram of the heavy-labeled mixed peptide in the LC-MS system. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] 1. Mixed heavy-labeled immune peptide sequence and heavy-labeled amino acid design

[0021] The present invention selects 10 MHCI immune peptides (see Table 1) as the constituent sequences of the mixed standard immune peptides based on the following criteria:

[0022] 1. It can cover the immune peptides bound by three types of molecules: HLAI (another name for human MHCI)-ABC;

[0023] 2. Contains the peptide segments with the highest frequency of HLA-A0201 and HLA-1101 molecules binding;

[0024] 3. Contains a classic and important neoantigen peptide sequence (KRAS-G12V);

[0025] 4. The peptide composition can cover the hydrophilic end, middle end and hydrophobic end of reverse phase chromatography.

[0026] Then, a non-terminal amino acid of these peptides is selected for re-labeling, and the re-labeled amino acids are mainly concentrated on leucine (Leucine, Leu, I), proline (Proline, Pro, P) and valine (Valine, Val, V) at specific sites.

[0027] During the solid phase synthesis of peptides, the corresponding carbon (13) nitrogen (15) fully weighted amino acids were added at the weighted positions to complete this round of synthesis. After weighted, the chromatographic properties of these peptides did not change, but a molecular weight shift of 6 Da (P, V) or 7 Da (L) occurred, which could be distinguished from non-weighted peptides in mass spectrometry and did not affect their retention properties in reversed phase chromatography (Table 1).

[0028] Table 1. Design of mixed heavy-labeled immune peptides

[0029] name Standard immune peptide HLA-Alleles Sequence mutation Hydrophilicity SEQ Neopep_RT1 ETHQQ{Val(13C5,15N)}VSR HLA-A*33:03 none Hydrophilic end NO.1 Neopep_RT2 EE{Val(13C5,15N)}KQTKRL HLA-B*44:03 none Hydrophilic end No.2 Neopep_RT3 VVVGADG{Val(13C5,15N)}GK HLA-A*11:01 G12V Hydrophilic end NO.3 Neopep_RT4 RPSG{Pro(13C5,15N)}GPEL HLA-A*02:01 none Hydrophilic end NO.4 Neopep_RT5 EEAQPI{Val(13C5,15N)}TKY HLA-B*18:01 none Middle End NO.5 Neopep_RT6 RLF{Val(13C5,15N)}GSIPK HLA-A*03:01 none Middle End No.6 Neopep_RT7 EE{Val(13C5,15N)}LIPDQKY HLA-B*44:02 none Middle End No.7 Neopep_RT8 KIADRF{Leu(13C6,15N)}LY HLA-C*12:03 none Hydrophobic end No.8 Neopep_RT9 QHFPAT{Pro(13C5,15N)}LL HLA-C*03:04 none Hydrophobic end No.9 Neopep_RT10 THALEW{Pro(13C5,15N)}SL HLA-B*15:10 none Hydrophobic end No.10

[0030] As shown in Table 1, the mixed heavy-labeled immune peptide design contains 10 human-derived immune peptides, including 10 common HLA typing-binding immune peptides and a classic new antigen mutation peptide, and can cover all areas of reversed-phase chromatography outflow, comprehensively considering all aspects of immune peptide detection quality control.

[0031] 2. Single-label sequence confirmation and mixing ratio optimization of heavy-labeled standard immune peptides

[0032] GeneScript was commissioned to synthesize the above immune peptide sequence as a standard heavy-label immune peptide, and the purity of each peptide segment was over 95%. On this basis, the amino acid sequence of the standard heavy-label immune peptide, as well as the confirmation of the heavy-label, and the optimization of the mixing ratio of the heavy-label immune peptide were completed, realizing the application of the mixed heavy-label standard immune peptide in the establishment of mass spectrometry methods and mixed standards.

[0033] The synthesized standard peptides were redissolved in 50% methanol and 50% double distilled water. The peptides had good solubility and could be completely dissolved. Subsequently, the single-labeled peptides were tested in a liquid-mass spectrometer to confirm that each peptide sequence could obtain a good secondary spectrum, and to obtain information such as the retention time of the mixed standard peptides, the best mass-to-charge ratio, and the mass spectrometry response ( Figure 1-10 ).

[0034] The specific method is: take 200fmol of peptide fragments through C18 reverse phase column separation, elute the peptide fragments with a short gradient, use targeted mass spectrometry detection, and obtain data for spectrum analysis using PEAKS ONLINE software. The present invention uses a set of high performance liquid chromatography (nEASY-nLC) equipped with Orbitrap Tribid Eclipse high-resolution mass spectrometry for single-label testing. Specific liquid phase and mass spectrometry parameters are shown in Tables 2 and 3. The data analysis platform uses BSI (Bioinformatics Solutions Inc.), and the company's PEAKS software analysis is obtained, and the results are shown in Table 4.

[0035] Table 2. Chromatographic parameters of the HPLC-MS method for single-label peptide detection

[0036] gradient: Time (mm:ss) Duration (mm:ss) Flow rate (nl / min) Mixture concentration (%B) 00:00 00:00 300 2 08:00 08:00 300 40 08:10 00:10 400 100 17:10 09:10 400 100 17:20 00:10 400 2 20:20 03:00 400 2

[0037] Table 3. Mass spectrometry parameters for single-label peptide detection using HPLC-MS / MS method

[0038]

[0039] Table 4. Data analysis parameters for single-labeled peptide detection

[0040]

[0041] In the early stage of single-label detection, it was found that the response of the hydrophobic end immune peptide was low. Therefore, when mixing the standard peptides, the molar ratio of the last three hydrophobic end immune peptides (RT8-10) was increased by 10 times to improve the mass spectrometry response and ensure that the mass spectrometry response of all immune peptides was within the appropriate range. The standard peptide mixing ratio and mass spectrometry response are shown in Table 5. A 3-30pmol / μL mixed standard peptide stock solution was prepared and named neo_pepmix. Neo_pepmix mixed standard peptides were loaded with 10-100 femtomoles and detected by Tims TOF pro2 (Bruker). The results are as follows Fig.11 As shown, all peptides have good chromatographic peak shape, good liquid-mass response and wide chromatographic retention range.

[0042] Table 5. Mixing ratio of heavy-labeled immune peptides (molar ratio)

[0043] name Standard immune peptide Mixing ratio Mass Spectrum Response* Neopep_RT1 ETHQQ{Val(13C5,15N)}VSR 1 2.59e5 Neopep_RT2 EE{Val(13C5,15N)}KQTKRL 1 3.01e5 Neopep_RT3 VVVGADG{Val(13C5,15N)}GK 1 1.76e5 Neopep_RT4 RPSG{Pro(13C5,15N)}GPEL 1 2.83e5 Neopep_RT5 EEAQPI{Val(13C5,15N)}TKY 1 3.15e5 Neopep_RT6 RLF{Val(13C5,15N)}GSIPK 1 2.24e5 Neopep_RT7 EE{Val(13C5,15N)}LIPDQKY 1 2.15e5 Neopep_RT8 KIADRF{Leu(13C6,15N)}LY 10 3.60e5 Neopep_RT9 QHFPAT{Pro(13C5,15N)}LL 10 3.49e5 Neopep_RT10 THALEW{Pro(13C5,15N)}SL 10 2.96e5

[0044] 3. Add the re-labeled standard immune peptide mixture neo_pepmix to the immune peptide sample for detection

[0045] In order to test the detection effect and influence of adding neo_pepmix to the measured samples. The present invention prepares 9 samples by mixing the neo_pepmix reserve solution with an equal amount of lymphoma cell line HLAI immune peptide samples according to different molar amounts (0.064, 0.16, 0.32, 0.8, 1.6, 4, 8, 20, 40 fetal mol), and uses our immune peptide detection method (DataDependentAcquisition mode) to collect LCMSMS data. This acquisition mode indiscriminately selects peptides with higher intensity for secondary identification, and can collect neo_pepmix peptide information while detecting lymphoma cell line HLAI immune peptides, which meets our investigation purpose and provides a reference for the subsequent application of neo_pepmix in actual samples. Mass spectrometry data analysis is performed using search parameters similar to those in Table 4, only the search database is changed to a merged library of standard peptides and human proteins to obtain a peptide identification list.

[0046] The experimental results of this part of the invention analyzed the total number of immune peptide identifications, the number of neo_pepmix member peptide identifications, and the number of neo_pepmix member peptide quantifications. The statistical results show that when the amount of mixed standard added is small (0.064-0.32fmol), the effect on HLAI immune peptide detection is small, but it is impossible to achieve full identification of heavy-standard peptides. When the amount of mixed standard added is large (such as 20f-40fmol), it will affect the depth of HLAI immune peptide identification. The addition of 0.8-8fmol of mixed heavy-standard peptide segments will not significantly affect the number of HLAI sample immune peptide group identifications, and can ensure that all 10 heavy-standard peptide segments of neo_pepmix are identified and quantified, meeting the needs of neo_pepmix as a quality control for actual detection of MHCI samples.

[0047] This indicates that an appropriate amount of re-labeled mixed standard immune peptides can be used to monitor the mass spectrometry method of actual MHCI immune peptide samples without significantly affecting the number of immune peptides detected in the actual samples.

[0048] Table 6. Peptide identification and quantitative statistics of different molar numbers of mixed standard peptides added to equal amounts of immune peptide group samples

[0049]

[0050] In Table 6, ISL represents neo_pepmix, in which different femtomolar weight-labeled immune peptides are added to an equal amount (equivalent to 4e6 human lymphoma cell line enrichment) of HLAI immune peptide samples; Peptide# is the total number of immune peptides identified; Mixed standard peptide identification# (identification number (total)): the number of mixed standard peptides identified among the total number of identified immune peptides, and the number in brackets is the total number of mixed standards; Mixed standard peptide quantification# (quantification number (total)): the number of mixed standard peptides quantified, and the number in brackets is the total number of mixed standards.

[0051] References:

[0052] Escobar, H., Reyes-Vargas, E., Jensen, PE, Delgado, JC, & Crockett, DK (2011). Utility of characteristic QTOF MS / MS fragmentation for MHC class ipeptides. Journal of Proteome Research, 10(5), 2494–2507.

[0053] Kote,S.,Pirog,A.,Bedran,G.,Alfaro,J.,&Dapic,I.(2020).Massspectrometry-based identification of MHC-associated peptides.In Cancers(Vol.12,Issue 3).MDPIAG.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A re-labeled standard MHC I immune peptide mixture, characterized in that: It includes 10 immune peptides, the amino acid sequences of which are shown in SEQ ID NO.1-10; one non-terminal amino acid in each immune peptide is 13 C5 and 15 N mark.

2. A re-labeled standard MHC I immune peptide mixture as claimed in claim 1, characterized in that: The labeled amino acids are leucine, proline, and valine.

3. A mixed standard heavy-labeled MHC I immune peptide according to claim 2, characterized in that: The valine at position 6 in the amino acid sequence shown in SEQ ID NO.1 was 13 C5 and 15 N mark; The valine at position 3 in the amino acid sequence shown in SEQ ID NO.2 was 13 C5 and 15 N mark; The valine at position 8 in the amino acid sequence shown in SEQ ID NO.3 was 13 C5 and 15 N mark; The proline at position 5 in the amino acid sequence shown in SEQ ID NO.4 was 13 C5 and 15 N mark; The valine at position 7 in the amino acid sequence shown in SEQ ID NO.5 was 13 C5 and 15 N mark; The valine at position 4 in the amino acid sequence shown in SEQ ID NO.6 was 13 C5 and 15 N mark; The valine at position 3 in the amino acid sequence shown in SEQ ID NO.7 was 13 C5 and 15 N mark; The leucine at position 7 in the amino acid sequence shown in SEQ ID NO.8 was 13 C5 and 15 N mark; The proline at position 7 in the amino acid sequence shown in SEQ ID NO.9 was 13 C5 and 15 N mark; The proline at position 7 in the amino acid sequence shown in SEQ ID NO.10 was 13 C5 and 15 N mark.

4. A mixed standard heavy-labeled MHCI immune peptide according to claim 4, characterized in that: The molar ratio of the mixture of the 10 immune peptides shown in SEQ ID NO.1-10 is 1:1:1:1:1:1:1:10:10:

10.

5. Use of a mixed standard re-labeled MHC I immune peptide segment as described in any one of claims 1 to 4 in HLA I immune peptide detection.

6. A HLAI immune peptide detection kit, characterized in that: The method comprises a HLA I immune peptide sample and a re-labeled standard MHC I immune peptide segment mixture as described in any one of claims 1 to 4.

7. A kit for detecting an immune peptide as claimed in claim 6, characterized in that: The amount of the re-labeled standard MHC I immune peptide mixture is 0.8-8fmol / μL.

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