A method for efficiently enriching a plurality of n-terminal modified peptide fragments of proteins

Abstract

The application provides a method for efficiently enriching N-terminal modified peptide segments of multiple proteins, comprising the following steps: 1) using an amino active reagent to specifically block the first free amino group of the side chain of lysine in a protein; 2) using a proteolytic enzyme to enzymatically digest the protein to generate N-terminal modified peptide segments and non-N-terminal modified peptide segments; 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 segments, so as to obtain labeled non-N-terminal modified peptide segments; and 4) using a reversed-phase chromatography stationary phase to separate the N-terminal modified peptide segments from the labeled non-N-terminal modified peptide segments, and enrich the N-terminal modified peptide segments; the method has the advantages that the side chain amino group of lysine in the protein is specifically blocked, a strong hydrophobic group is introduced into the non-N-terminal modified peptide segments generated after enzymatic digestion, and the non-N-terminal modified peptide segments are efficiently removed in combination with reversed-phase chromatography separation, so that the N-terminal modified peptide segments in a complex biological sample can be selectively and non-discriminately enriched.

Description

Technical Field

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

[0002] N-terminal modifications of proteins refer to post-translational modifications that occur on the N-terminal amino acid sequence after protein synthesis. Common N-terminal modifications include acetylation, methylation, cyclization, fatty acid acylation, and protease hydrolysis. Large-scale analysis of N-terminal modifications at the proteomic level helps to comprehensively understand the roles of N-terminal modifications in various physiological and pathological processes. For example, acetylation, as the most common post-translational modification at the N-terminus of proteins, plays an important role in regulating protein function, stability, interactions, and subcellular localization. Due to the complex composition and wide abundance dynamic range of proteins in biological samples, and the low abundance of N-terminal modified peptides after enzyme digestion, efficient enrichment of N-terminal modified peptides is necessary before mass spectrometry analysis to improve the identification coverage of N-terminal modified peptides.

[0003] Currently, enrichment methods for N-terminal modified peptides of proteins are mainly divided into two categories. One approach utilizes the charge difference between N-terminal modified peptides and non-N-terminal modified peptides after protein digestion, employing strong cation exchange chromatography to separate and enrich N-terminal modified peptides (Mol. Cell. Proteomics 2021, 20, 100003; J. Proteome Res. 2013, 12, 3277-3287.). However, this method cannot efficiently separate histidine-containing N-terminal modified peptides from non-N-terminal modified peptides, resulting in the loss of N-terminal modified peptides and affecting enrichment efficiency and accuracy. Another approach leverages the exposure of new α-amino groups at the N-terminus of non-N-terminal modified peptides after protein digestion, using amino-active materials to capture and remove these peptides, thus achieving reverse enrichment of N-terminal modified peptides (Anal. Chem. 2020, 92, 8315-8322; J. Proteome Res. 2013, 12, 3277-3287.). (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. Furthermore, the introduction of a large number of functional materials leads to the loss of N-terminal modified peptides due to non-specific adsorption, thus limiting the enrichment selectivity of this type of method.

[0004] To address the issue of insufficient solid-liquid reaction efficiency in reverse enrichment methods, the previously reported authorized patent CN107305172B discloses "A Protein N-Terminal Enrichment Method Based on Hydrophobic Group Modification." This method leverages the high efficiency of liquid-liquid reactions by introducing hydrophobic groups onto the α-amino groups of non-N-terminal peptides using an amino-active reagent. Based on the strong interaction between the hydrophobic groups and C18 reversed-phase chromatography, non-N-terminal peptides are easily and rapidly removed during conventional sample desalting, thus achieving efficient enrichment of N-terminal peptides. However, this method enriches not only naturally occurring N-terminal modified peptides but also a large number of unmodified N-terminal peptides, severely interfering with the enrichment and detection of N-terminal modified peptides. It 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 non-discrimination against N-terminal modified peptides with different properties.

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

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

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

[0009] 2) The protein is hydrolyzed using a proteolytic enzyme to produce N-terminal modified peptides and non-N-terminal modified peptides;

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

[0011] 4) The N-terminal modified peptide and the labeled non-N-terminal modified peptide are separated by reversed-phase chromatography stationary phase, and the N-terminal modified peptide is enriched.

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

[0013] (1) The present invention uses specific amino-active reagents 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, thus avoiding the N-terminal modified peptide containing lysine being labeled with hydrophobic groups in subsequent experiments, and effectively improving the recovery rate of N-terminal modified peptide.

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

[0015] (3) The high efficiency of hydrophobic group labeling in this invention, and the strong hydrophobic interaction between the reversed-phase chromatographic stationary phase and the non-N-terminal modified peptides labeled with hydrophobic groups, cause a significant change in the chromatographic retention time of the peptides, which promotes the efficient separation of N-terminal modified peptides and non-N-terminal modified peptides. Among them, N-terminal non-modified peptides can also be separated, thereby achieving highly selective enrichment of N-terminal modified peptides.

[0016] (4) The reversed-phase chromatographic stationary phase of the present invention does not have non-specific adsorption of N-terminal modified peptides with different properties, thus avoiding the loss of N-terminal modified peptides and achieving non-discriminatory enrichment of multiple N-terminal modified peptides.

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

[0018] Specifically, the amino-active reagent described in step 1) is used for the specific blocking under alkaline conditions with pH ≥ 11.

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

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

[0021] Specifically, 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℃, the blocking is carried out in an alkaline reagent with a pH 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 amino-active reagent with a carbon chain described in step 3) has a carbon chain length of 8-20 and contains an aldehyde group or a succinimide ester group, with the following specific structure:

[0025]

[0026] Where n = 3 - 9.

[0027] Specifically, the N-terminal modifications of the various proteins are one or more of the following: 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 the following: cells, tissues, and body fluids. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the enrichment process of the N-terminal modified peptides of the present invention.

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

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

[0031] Figure 3 (a) represents the proportions of N-terminal modified peptides and non-N-terminal modified peptides in the HeLa cell enzymatic hydrolysis peptides before enrichment in Example 2.

[0032] Figure 3 (b) represents the proportions of N-terminal modified peptides and non-N-terminal modified peptides in the enzymatically hydrolyzed peptides of HeLa cells enriched in Example 2. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0034] Figure 1 This is a flowchart illustrating the enrichment process of N-terminal modified peptides in proteins according to the present invention. After denaturation and reductive alkylation, the protein sample is specifically blocked using a specific amino-active reagent against the first free amino group (K) of the lysine side chain. Figure 1 The process involves: first, using the I part of the protein, without blocking the free amino groups at the N-terminus; then, enzymatically digesting the protein with trypsin to produce N-terminal modified peptides and non-N-terminal modified peptides without free amino groups; finally, using an amino-active reagent with a carbon chain as a strongly 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 unmodified peptides (…). Figure 1The third free amino group in the non-N-terminal peptide (part II) and non-N-terminal peptide ( Figure 1 Part III of the study utilizes the hydrophobicity difference between N-terminal modified peptides and non-N-terminal modified peptides, employing reversed-phase chromatography stationary phase to efficiently separate N-terminal modified peptides from labeled non-N-terminal modified peptides, thereby achieving simultaneous enrichment of multiple N-terminal modified peptides.

[0035] This invention specifically blocks the side chain amino groups of lysine in proteins and introduces strongly hydrophobic groups into non-N-terminal modified peptides generated after enzymatic hydrolysis using a simple and efficient labeling method. Then, it combines reversed-phase chromatography to efficiently remove non-N-terminal modified peptides. In addition, N-terminal unmodified peptides can also be efficiently separated and removed, thereby achieving highly selective and non-discriminatory enrichment of N-terminal modified peptides in complex biological samples.

[0036] Example 1

[0037] 1.1 First, dissolve 100 μg of bovine serum albumin (BSA) in 100 μL of 100 mM phosphate buffer (pH 8) containing 1% sodium deoxycholate, and denature at 90 °C for 5 min; 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.

[0038] Then, add O-methylisourea hydrogen sulfate to a final concentration of 1000 mM, adjust the pH to 11, react at 37 °C for 2 h, and then transfer the BSA sample to an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain the first solution.

[0039] 1.2 High-speed centrifugation was used to remove excess reagent from the first solution in step 1.1. After washing the filter membrane with 100 mM phosphate buffer (pH 8), trypsin was added for enzymatic hydrolysis, with the amount of trypsin being 1 / 50 (w / w) of the BSA mass. Enzymatic hydrolysis was carried out at 37°C for 12 h. The BSA-hydrolyzed peptides were collected by high-speed centrifugation and mixed with a standard N-terminal acetylated peptide (sequence Ac-KRGGGGYIKIIKV) at a mass ratio of 100:1. The mixture was then freeze-dried to obtain the processed 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-dioxopyrrolidone-1-yldodecanoate and reacted at 40 °C for 4 h to obtain the second solution.

[0041] 1.4 After lyophilizing the second solution from 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). Load the 2% phase B solution onto a C18 reversed-phase column (4.6 mm id × 15 cm, packing particle size 5 μm). Separate and elute peptides using a stepwise gradient of 2-30-90% phase B within 30 min. Collect the N-terminal acetylated peptide fraction, lyophilize it, and obtain the enriched sample b. Perform MALDI-TOF mass spectrometry analysis on samples a and b respectively. The results are 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 peptide mixtures.

[0042] Example 2

[0043] 2.1 Using HeLa cells as the actual biological sample. First, HeLa cells were suspended in 100 μL of 100 mM phosphate buffer (pH 8) containing 1% sodium deoxycholate. After sonication to disrupt the cells, the extracted proteins were denatured at 90°C for 5 min.

[0044] Add 5 μL of 100 mM dithiothreitol and 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 with a final concentration of 1500 mM was added, 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 the first solution.

[0046] 2.2 High-speed centrifugation was used to remove excess reagents from the first solution in step 2.1. After washing the filter membrane with 100mM phosphate buffer at pH 8, trypsin was added for enzymatic hydrolysis. The amount of trypsin used was 1 / 50 (w / w) of the HeLa cell sample mass. The hydrolysis was carried out at 37℃ for 12h. The hydrolyzed peptides were collected by high-speed centrifugation and freeze-dried to obtain the processed HeLa cell hydrolyzed peptide sample a.

[0047] 2.3 The treated HeLa cell enzymatically hydrolyzed peptides were redissolved in 100 μL of phosphate buffer (pH 8) containing 100 mM 2,5-dioxopyrrolidone-1-yldodecanoate and reacted at 40 °C for 4 h to obtain the second solution.

[0048] 2.4 After lyophilizing the second solution from 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). Load the 2% phase B solution onto a C18 reversed-phase column (4.6 mm id × 15 cm, packing particle size 5 μm). Separate and elute peptides using a stepwise gradient of 2-40-90% phase B within 30 min. Collect the N-terminal modified peptide fraction, lyophilize it, and obtain the enriched sample b. Perform nanoRPLC-ESI-MS / MS analysis and data processing on samples a and b, respectively. The results are as follows: Figure 3 (a) and Figure 3 As shown in (b). Figure 3 (a) shows the proportions of N-terminal modified peptides and non-N-terminal modified peptides in the HeLa cell enzymatic digestion peptides before enrichment. The proportion of non-N-terminal modified peptides is as high as 98%, while the proportion of N-terminal modified peptides is only 2%, and only acetylation modification was detected. Figure 3 (b) shows the proportions of N-terminal modified peptides and non-N-terminal modified peptides in the enriched HeLa cell enzymatic digestion peptides. The proportion of N-terminal modified peptides was as high as 89%, a significant increase of 45-fold (89% vs. 2%) compared to before enrichment. The detected N-terminal modified peptides included acetylation and cyclization modifications. The above analysis demonstrates that this method successfully achieves simultaneous and efficient enrichment of multiple N-terminal modifications in real biological samples.

[0049] Example 3

[0050] The rest is the same as in Example 2, except that:

[0051] The phrase "Add O-methylisourea hemisulfate to a final concentration of 1500 mM, adjust the pH to 11, react at 37°C for 2 h, and then transfer the protein sample to an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain the first solution" in step 2.1 is revised to "Add O-methylisourea hydrogen sulfate to a final concentration of 2500 mM, adjust the pH to 12, react at 37°C for 5 h, and then transfer the protein sample to an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain the first solution".

[0052] The phrase "redissolve the treated HeLa cell enzymatically hydrolyzed peptides in 100 μL of phosphate buffer (pH 8) containing 100 mM 2,5-dioxopyrrolidone-1-yldodecanoate and react at 40 °C for 4 h to obtain the second solution" in step 2.3 is revised to "redissolve the treated HeLa cell enzymatically hydrolyzed peptides in 100 μL of aqueous solution containing 50 mM hexadecaldehyde and 500 mM sodium cyanoborohydride and react at 50 °C for 4 h to obtain the second solution".

[0053] Modify "within 30 minutes, follow a step gradient of 2-40-90%B" in step 2.4 to "within 30 minutes, follow a step gradient of 2-50-90%B".

[0054] Example 4

[0055] The rest is the same as in Example 2, except that:

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

[0057] Modify "phosphate buffer containing 100mM 2,5-dioxopyrrolidone-1-yldodecanoate (pH 8)" in step 2.3 to "phosphate buffer containing 100mM N-hydroxysuccinimide palmitate (pH 8)".

[0058] Example 5

[0059] The rest is the same as in Example 2, except that:

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

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

[0062] Example 6

[0063] The rest is the same as in Example 2, except that:

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

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

[0066] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for efficiently enriching a plurality of N-terminal modified peptides, characterized in that include: 1) Use an amino-active reagent to specifically block the first free amino group of the lysine side chain in the protein; 2) The protein is hydrolyzed using a proteolytic enzyme to produce N-terminal modified peptides and non-N-terminal modified peptides; 3) The second and third free amino groups in the non-N-terminal modified peptide are labeled with an amino-active reagent with a carbon chain as a strong hydrophobic group to obtain the labeled non-N-terminal modified peptide. 4) The N-terminal modified peptide and the labeled non-N-terminal modified peptide are separated by reversed-phase chromatography stationary phase, and the N-terminal modified peptide is enriched. The specific blocking is performed using the amino-active reagent under alkaline conditions with pH ≥ 11.

2. The method for efficiently enriching multiple N-terminal modified peptides of proteins according to claim 1, characterized in that... The amino-active reagent is a guanidine-modifying reagent.

3. The method for efficiently enriching multiple N-terminal modified peptides of proteins according to claim 2, characterized in that... The guanidinizing agent is one or more of O-methylisourea hydrogen sulfate, O-methylisourea hemisulfate, aminocyanide, pyrazole-1-carboxamide, S-methyl-thiourea and their derivatives.

4. The method for efficiently enriching multiple N-terminal modified peptides of proteins according to claim 3, 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℃, the blocking is carried out in an alkaline reagent with a pH of 11-14, and the reaction time is 0.5-48 h.

5. The method for efficiently enriching multiple N-terminal modified peptides of proteins according to claim 4, characterized in that... The alkaline reagent with a pH value of 11-14 is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and ammonia water.

6. The method for efficiently enriching multiple N-terminal modified peptides of proteins according to claim 5, characterized in that... The final concentration of the protein is 1 / 10 to 1000 / 1 of the final concentration of the amino-active reagent.

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

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

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