Method for efficiently enriching N-terminal modified peptide fragments of multiple proteins

Abstract

The invention provides a method for efficiently enriching N-terminal modified peptide fragments of various proteins. The method comprises the following steps: 1) specifically closing first free amino groups of lysine side chains in the proteins by adopting an amino active reagent; (2) carrying out enzymolysis on the protein by adopting proteolytic enzyme to generate an N-terminal modified peptide fragment and a non-N-terminal modified peptide fragment; 3) marking a second free amino group and a third free amino group in the non-N-terminal modified peptide fragment by adopting an amino active reagent with a carbon chain as a strong hydrophobic group to obtain a marked non-N-terminal modified peptide fragment; (4) separating the N-terminal modified peptide fragment from the labeled non-N-terminal modified peptide fragment by adopting a reversed-phase chromatography stationary phase, and enriching the N-terminal modified peptide fragment; the method has the advantages that the side chain amino group of lysine in the protein is specifically closed, the strong hydrophobic group is introduced into the non-N-terminal modified peptide fragment generated after enzymolysis, and the non-N-terminal modified peptide fragment is efficiently removed in combination with reversed-phase chromatographic separation, so that high-selectivity and undiscriminated enrichment of the N-terminal modified peptide fragment in the complex biological sample is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of protein N-terminal peptide enrichment methods, and in particular relates to a method for efficiently enriching multiple protein N-terminal modified peptides. Background Art

[0002] N-terminal modification of proteins refers to the post-translational modification of the N-terminal amino acid sequence of proteins after synthesis. Common N-terminal modifications include acetylation, methylation, cyclization, fatty acylation, and protease hydrolysis. Large-scale analysis of N-terminal modification of proteins at the proteome level helps to fully understand the role of N-terminal modification in different physiological and pathological processes. For example, acetylation modification, as the most common post-translational modification of protein N-terminus, plays an important role in regulating protein function, stability, interaction, and subcellular localization. Due to the complex composition of proteins in biological samples, the wide dynamic range of abundance, and the low abundance of N-terminal modified peptides after enzyme cleavage, it is necessary to efficiently enrich the peptides containing N-terminal modifications before mass spectrometry analysis to improve the identification coverage of protein N-terminal modified peptides.

[0003] Currently, the enrichment methods for protein N-terminal modified peptides are mainly divided into two categories. One type is based on the difference in charge between the N-terminal modified peptides and the non-N-terminal modified peptides after protein hydrolysis, and uses strong cation exchange chromatography to separate and enrich the N-terminal modified peptides (Mol. Cell. Proteomics 2021, 20, 100003; J. Proteome Res. 2013, 12, 3277-3287.). However, the enrichment method based on strong cation exchange chromatography cannot efficiently separate the N-terminal modified peptides containing histidine from the non-N-terminal modified peptides, resulting in the loss of the N-terminal modified peptides, affecting the enrichment efficiency and accuracy; the other type is based on the exposure of new α-amino groups at the N-terminus of the non-N-terminal modified peptides after protein hydrolysis, and uses amino-active materials to capture and remove the non-N-terminal modified peptides, thereby achieving reverse enrichment of the N-terminal modified peptides (Anal. Chem. 2020, 92, 8315-8322; J. Proteome Res.2013,12,3277-3287.), however, the solid-liquid reaction efficiency of this type of method is often insufficient, which limits the capture efficiency of amino-active materials for non-N-terminal modified peptides. In addition, the introduction of a large amount of functional materials leads to the loss of N-terminal modified peptides due to nonspecific adsorption, which limits the enrichment selectivity of this type of method.

[0004] In response to the problem of insufficient solid-liquid reaction efficiency in reverse enrichment methods, the reported authorized patent CN107305172B discloses "a method for enriching the N-terminus of proteins based on hydrophobic group modification", which takes advantage of the high efficiency of liquid-liquid reaction and uses amino-active reagents to introduce hydrophobic groups on the α-amino groups of non-N-terminal peptides. Based on the strong interaction between hydrophobic groups and C18 reverse phase chromatography, non-N-terminal peptides are simply and quickly removed during the conventional sample desalting process, thereby achieving efficient enrichment of N-terminal peptides. However, in addition to natural N-terminal modified peptides, the N-terminal peptides enriched by this method also contain a large number of N-terminal non-modified peptides, which seriously interfere with the enrichment and detection of N-terminal modified peptides, and cannot selectively enrich N-terminal modified peptides, limiting its further application. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for efficiently enriching N-terminal modified peptides of various proteins, which has the advantages of high labeling efficiency, high enrichment selectivity, and no discrimination against N-terminal modified peptides of different properties.

[0006] The technical solution adopted by the present invention to solve the above technical problems is:

[0007] A method for efficiently enriching N-terminal modified peptides of various proteins, comprising:

[0008] 1) Using an amino-active reagent to specifically block the first free amino group of the lysine side chain in the protein;

[0009] 2) using a proteolytic enzyme to enzymatically hydrolyze the protein to produce an N-terminal modified peptide segment and a non-N-terminal modified peptide segment;

[0010] 3) using an amino-active reagent with a carbon chain as a strong hydrophobic group to label the second free amino group and the third free amino group in the non-N-terminal modified peptide segment to obtain a labeled non-N-terminal modified peptide segment;

[0011] 4) Using a reverse phase chromatographic stationary phase to separate the N-terminal modified peptide segment and the labeled non-N-terminal modified peptide segment, and enriching the N-terminal modified peptide segment.

[0012] Compared with the prior art, the advantages of the present invention are:

[0013] (1) The present invention uses a specific amino-active reagent and specific blocking conditions to specifically block the first free amino group of the lysine side chain without blocking the free amino group at the N-terminus of the protein, thereby avoiding the N-terminal modified peptide containing lysine being labeled with a hydrophobic group in subsequent experiments, and effectively improving the recovery rate of the N-terminal modified peptide.

[0014] (2) The blocked lysine of the present invention can not only be hydrolyzed by trypsin, so that the length of the generated N-terminal modified peptide segment is conducive to mass spectrometry detection; it can also significantly improve the ionization efficiency of the N-terminal modified peptide segment in the mass spectrometry, thereby improving the identification coverage of the N-terminal modified peptide segment.

[0015] (3) The high efficiency of the hydrophobic group labeling of the present invention and the strong hydrophobic interaction between the reverse phase chromatographic stationary phase and the non-N-terminal modified peptides labeled with the hydrophobic group cause a significant change in the chromatographic retention time of the peptides, thereby promoting the efficient separation of the N-terminal modified peptides from the non-N-terminal modified peptides, wherein the N-terminal non-modified peptides can also be separated together, thereby achieving highly selective enrichment of the N-terminal modified peptides.

[0016] (4) The reverse phase chromatography stationary phase of the present invention has no non-specific adsorption to N-terminal modified peptides of different properties, thereby avoiding the loss of N-terminal modified peptides, thereby achieving non-discriminatory enrichment of multiple N-terminal modified peptides.

[0017] (5) The present invention does not require the introduction of a large amount of affinity materials and additional sample purification processes, has fewer enrichment steps, and a simple process, which effectively reduces sample loss and can achieve simultaneous and efficient enrichment of multiple N-terminal modified peptides.

[0018] Specifically, the amino-active reagent in step 1) is subjected to the specific blocking under alkaline conditions of pH ≥ 11.

[0019] Specifically, the amino-active reagent is a guanidinating reagent.

[0020] Specifically, the guanidinating agent is one or more of O-methylisourea hydrogen sulfate, O-methylisourea hemisulfate, cyanamide, pyrazole-1-carboxamide, S-methyl-thiourea and their derivatives.

[0021] Specifically, the specific blocking conditions are: the final concentration of the amino-active reagent used for blocking is 100-4000 mM, the temperature is 4-70° C., the blocking is carried out in an alkaline reagent with a pH value of 11-14, and the reaction time is 0.5-48 h.

[0022] Specifically, the alkaline reagent with a pH value of 11 to 14 is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and ammonia water.

[0023] Specifically, the final concentration of the protein is 1 / 10 to 1000 / 1 of the final concentration of the amino-active reagent.

[0024] Specifically, the carbon chain length of the amino-active reagent with a carbon chain in step 3) is 8-20, and contains an aldehyde group or a succinimide ester group, and the specific structure is as follows:

[0025]

[0026] Among them, n=3-9.

[0027] Specifically, the N-terminal modifications of the plurality of proteins are one or more of acetylation, formylation, methylation, amidation, cyclization, fatty acylation, glycylation, protease hydrolysis or methionine removal; the proteins are proteins extracted from biological samples, and the biological samples are one or more of cells, tissues, and body fluids. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a flow chart of the enrichment of the N-terminal modified peptide segment of the present invention;

[0029] Figure 2 (a) is a MALDI-TOF mass spectrum of the N-terminal acetylated peptide (*, mass-to-charge ratio m / z 1557) in Example 1 mixed with the trypsin hydrolyzate of bovine serum albumin at a mass ratio of 1:100;

[0030] Figure 2 (b) is a MALDI-TOF mass spectrum of the N-terminal acetylated modified peptide in Example 1 and the trypsin hydrolyzate of bovine serum albumin mixed at a mass ratio of 1:100 and enriched;

[0031] Figure 3 (a) is the ratio of the N-terminal modified peptide segment and the non-N-terminal modified peptide segment in the HeLa cell enzymatic peptide segment before enrichment in Example 2;

[0032] Figure 3 (b) is the respective proportions of N-terminal modified peptides and non-N-terminal modified peptides in the HeLa cell enzymatic peptides enriched in Example 2. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the accompanying drawings.

[0034] Figure 1 The enrichment flow chart of the protein N-terminal modified peptide segment of the present invention. After the protein sample is denatured, reduced and alkylated, a specific amino-active reagent is used to specifically block the first free amino group of the lysine (K) side chain ( Figure 1 The I part of the protein) is not blocked in the free amino group of the N-terminus of the protein; the protein is hydrolyzed with trypsin to produce N-terminal modified peptides and non-N-terminal modified peptides without free amino groups; and an amino-active reagent with a carbon chain is used as a strong hydrophobic group to label the free amino groups in the non-N-terminal modified peptides, including the second free amino group in the N-terminal non-modified peptides ( Figure 1The second part of the peptide) and the third free amino group in the non-N-terminal peptide ( Figure 1 Part III of the above); utilizing the difference in hydrophobicity between the N-terminal modified peptide and the non-N-terminal modified peptide, a reversed phase chromatographic stationary phase is used to efficiently separate the N-terminal modified peptide and the labeled non-N-terminal modified peptide, thereby achieving simultaneous enrichment of multiple N-terminal modified peptides.

[0035] The present invention specifically blocks the side chain amino group of lysine in the protein, and adopts a simple and efficient labeling method to introduce a strong hydrophobic group into the non-N-terminal modified peptide segment produced after enzymatic hydrolysis, and then combines the reverse phase chromatography separation to efficiently remove the non-N-terminal modified peptide segment, wherein the N-terminal non-modified peptide segment can also be efficiently separated and removed, thereby achieving highly selective and non-discriminatory enrichment of N-terminal modified peptide segments in complex biological samples.

[0036] Embodiment 1

[0037] 1.1 First, 100 μg of bovine serum albumin (BSA) was dissolved in 100 μL of 100 mM phosphate buffer (pH 8) containing 1% sodium deoxycholate, and denatured at 90°C for 5 min; 5 μL of 100 mM dithiothreitol was added, and the mixture was reacted at 56°C for 1 h, and then 2 μL of 500 mM iodoacetamide was added, and the mixture was reacted at room temperature in the dark for 30 min;

[0038] Then, O-methylisourea hydrogen sulfate was added to a final concentration of 1000 mM, and the pH was adjusted to 11. After reacting at 37° C. for 2 h, the BSA sample was transferred to an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a first solution.

[0039] 1.2 High-speed centrifugation was used to remove excess reagents in the first solution of step 1.1, and 100 mM phosphate buffer with a pH of 8 was added to wash the filter membrane, followed by addition of trypsin for enzymatic hydrolysis, wherein the amount of trypsin used was 1 / 50 (w / w) of the mass of BSA, and the enzymatic hydrolysis was performed at 37°C for 12 h. The BSA enzymatic peptides were collected by high-speed centrifugation, mixed with the guanidinized standard N-terminal acetylated modified peptide (sequence: Ac-KRGGGGYIKIIKV) at a mass ratio of 100:1, and freeze-dried to obtain the treated peptide mixture sample a.

[0040] 1.3 The treated peptide mixture was redissolved in 100 μL of phosphate buffer (pH 8) containing 10 mM 2,5-dioxopyrrolidin-1-yl dodecanoate, and reacted at 40° C. for 4 h to obtain a second solution.

[0041] 1.4 After freeze-drying the second solution in step 1.3, redissolve it in 5% (v / v) phase B (phase A, 2% acetonitrile + 0.1% trifluoroacetic acid aqueous solution; phase B, 98% acetonitrile + 0.1% trifluoroacetic acid aqueous solution), use 2% phase B to load on C18 reverse phase separation column (4.6mm id×15cm, filler particle size 5μm), within 30min, follow the step gradient 2-30-90% B, separate and elute the peptides, collect the N-terminal acetylated peptide fraction, freeze-dry, and obtain the enriched sample b. MALDI-TOF mass spectrometry analysis was performed on samples a and b, and the results were as follows: Figure 2 (a) and Figure 2 As shown in (b), MALDI-TOF mass spectrometry analysis confirmed that N-terminal acetylated peptides can be efficiently and selectively enriched from a peptide mixture.

[0042] Embodiment 2

[0043] 2.1 HeLa cells were used as the actual biological sample. First, HeLa cells were suspended in 100 μL of 100 mM pH 8 phosphate buffer containing 1% sodium deoxycholate. After the cells were disrupted by ultrasound, the extracted proteins were denatured at high temperature at 90°C for 5 min.

[0044] Add 5 μL of 100 mM dithiothreitol, react at 56°C for 1 h, then add 2 μL of 500 mM iodoacetamide, and react at room temperature in the dark for 30 min;

[0045] Then, O-methylisourea hemisulfate was added to a final concentration of 1500 mM, and the pH was adjusted to 11. After reacting at 37° C. for 2 h, the protein sample was transferred to an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a first solution.

[0046] 2.2 Remove excess reagent in the first solution of step 2.1 by high-speed centrifugation, add 100 mM phosphate buffer with a pH of 8 to wash the filter membrane, and then add trypsin for enzymatic hydrolysis, wherein the amount of trypsin used is 1 / 50 (w / w) of the mass of the HeLa cell sample, and enzymatic hydrolysis is carried out at 37°C for 12 hours; collect the enzymatic peptides by high-speed centrifugation and freeze-dry them to obtain the treated HeLa cell enzymatic peptide sample a.

[0047] 2.3 The treated HeLa cell enzymatic peptide fragments were redissolved in 100 μL of phosphate buffer (pH 8) containing 100 mM 2,5-dioxopyrrolidino-1-yl dodecanoate, and reacted at 40° C. for 4 h to obtain a second solution.

[0048] 2.4 After freeze-drying the second solution in step 2.3, redissolve it in 5% (v / v) phase B (phase A, 2% acetonitrile + 0.1% trifluoroacetic acid aqueous solution; phase B, 98% acetonitrile + 0.1% trifluoroacetic acid aqueous solution), use 2% phase B to load on C18 reverse phase separation column (4.6mm id×15cm, filler particle size 5μm), within 30min, follow the step gradient 2-40-90% B, separate and elute the peptides, collect the N-terminal modified peptide fraction, freeze-dry, and obtain the enriched sample b. NanoRPLC-ESI-MS / MS analysis and data processing were performed on samples a and b, respectively. The results are shown in Figure 3 (a) and Figure 3 (b) Figure 3 (a) The proportions of N-terminal modified peptides and non-N-terminal modified peptides in the HeLa cell enzymatic peptides before enrichment, among which non-N-terminal modified peptides accounted for as high as 98%, N-terminal modified peptides accounted for only 2%, and only acetylation modification was detected; Figure 3 (b) is the proportion of N-terminal modified peptides and non-N-terminal modified peptides in the enriched HeLa cell enzymatic peptides, among which the proportion of N-terminal modified peptides is as high as 89%, which is significantly increased by 45 times compared with before enrichment (89% vs 2%). The detected N-terminal modified peptides include acetylation modification, cyclization modification, etc. The above analysis shows that this method successfully achieves the simultaneous and efficient enrichment of multiple N-terminal modifications in actual biological samples.

[0049] Embodiment 3

[0050] The rest is the same as the second embodiment, except that:

[0051] Modify step 2.1 from "add O-methylisourea hemisulfate to a final concentration of 1500 mM, adjust the pH to 11, react at 37°C for 2 h, and transfer the protein sample to an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a first solution" to "add O-methylisourea hydrogensulfate to a final concentration of 2500 mM, adjust the pH to 12, react at 37°C for 5 h, and transfer the protein sample to an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain a first solution";

[0052] Modify step 2.3 from "redissolve the treated HeLa cell enzymatic peptide fragments in 100 μL of phosphate buffer (pH 8) containing 100 mM 2,5-dioxopyrrolidin-1-yl dodecanoate, react at 40°C for 4 h to obtain a second solution" to "redissolve the treated HeLa cell enzymatic peptide fragments in 100 μL of an aqueous solution containing 50 mM hexadecanal and 500 mM sodium cyanoborohydride, react at 50°C for 4 h to obtain a second solution";

[0053] Modify step 2.4 from “2-40-90% B in 30 min by step gradient” to “2-50-90% B in 30 min by step gradient”.

[0054] Embodiment 4

[0055] The rest is the same as the second embodiment, except that:

[0056] Change "HeLa cells" in step 2.1 to "yeast cells";

[0057] Modify the “phosphate buffer (pH 8) containing 100 mM 2,5-dioxopyrrolidin-1-yl dodecanoate” in step 2.3 to “phosphate buffer (pH 8) containing 100 mM palmitic acid N-hydroxysuccinimide ester”.

[0058] Embodiment 5

[0059] The rest is the same as the second embodiment, except that:

[0060] Change "HeLa cells" in step 2.1 to "yeast cells";

[0061] Modify step 2.1 from "add O-methylisourea hemisulfate at a final concentration of 1500mM, adjust the pH to 11, react at 37°C for 2h, and transfer the protein sample to an ultrafiltration membrane with a molecular weight cutoff of 10kDa to obtain a first solution" to "add cyanamide reagent at a final concentration of 4000mM, adjust the pH to 14, react at 55°C for 10h, and transfer the protein sample to an ultrafiltration membrane with a molecular weight cutoff of 20kDa to obtain a first solution".

[0062] Embodiment 6

[0063] The rest is the same as the second embodiment, except that:

[0064] Change "HeLa cells" in step 2.1 to "serum";

[0065] Modify step 2.1 from "add O-methylisourea hemisulfate to a final concentration of 1500mM, adjust the pH to 11, react at 37°C for 2h, and then transfer the protein sample to an ultrafiltration membrane with a molecular weight cutoff of 10kDa to obtain a first solution" to "add O-methylisourea hemisulfate to a final concentration of 1000mM and 1000mM cyanamide, adjust the pH to 12, react at 45°C for 4h, and then transfer the protein sample to an ultrafiltration membrane with a molecular weight cutoff of 30kDa to obtain a first solution".

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for efficiently enriching N-terminal modified peptides of various proteins, characterized in that include: 1) Using an amino-active reagent to specifically block the first free amino group of the lysine side chain in the protein; 2) using a proteolytic enzyme to enzymatically hydrolyze the protein to produce an N-terminal modified peptide segment and a non-N-terminal modified peptide segment; 3) using an amino-active reagent with a carbon chain as a strong hydrophobic group to label the second free amino group and the third free amino group in the non-N-terminal modified peptide segment to obtain a labeled non-N-terminal modified peptide segment; 4) Using a reverse phase chromatographic stationary phase to separate the N-terminal modified peptide segment and the labeled non-N-terminal modified peptide segment, and enriching the N-terminal modified peptide segment.

2. A method for efficiently enriching multiple protein N-terminal modified peptides according to claim 1, characterized in that The amino-active reagent in step 1) is subjected to the specific blocking under alkaline conditions of pH ≥ 11.

3. A method for efficiently enriching multiple protein N-terminal modified peptides according to claim 2, characterized in that The amino-active reagent is a guanidinating reagent.

4. A method for efficiently enriching multiple protein N-terminal modified peptides according to claim 3, characterized in that The guanidinating agent is one or more than two of O-methylisourea hydrogen sulfate, O-methylisourea hemisulfate, cyanamide, pyrazole-1-carboxamide, S-methyl-thiourea and their derivatives.

5. A method for efficiently enriching multiple protein N-terminal modified peptides according to claim 2, characterized in that The specific blocking conditions are as follows: the final concentration of the amino-active reagent used for blocking is 100-4000 mM, the temperature is 4-70° C., the blocking is carried out in an alkaline reagent with a pH value of 11-14, and the reaction time is 0.5-48 h.

6. A method for efficiently enriching multiple protein N-terminal modified peptides according to claim 5, characterized in that The alkaline reagent with a pH value of 11 to 14 is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate and ammonia water.

7. A method for efficiently enriching multiple protein N-terminal modified peptides according to claim 6, characterized in that The final concentration of the protein is 1 / 10 to 1000 / 1 of the final concentration of the amino-active reagent.

8. A method for efficiently enriching multiple protein N-terminal modified peptides according to claim 1, characterized in that The carbon chain length of the amino-active reagent with a carbon chain in step 3) is 8-20, and contains an aldehyde group or a succinimide ester group. The specific structural formula is as follows: Among them, n=3-9.

9. A method for efficiently enriching multiple protein N-terminal modified peptides according to claim 1, characterized in that The N-terminal modifications of the various proteins are one or more of acetylation, formylation, methylation, amidation, cyclization, fatty acylation, glycylation, protease hydrolysis or methionine removal; the proteins are proteins extracted from biological samples, and the biological samples are one or more of cells, tissues, and body fluids.

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