CN112980908B - SUMO peptide fragment enrichment method based on SUMO enzyme and SAX removal - Google Patents

Abstract

The invention relates to an enrichment method of SUMO modified peptide segments based on SUMO removal enzyme and anion exchange chromatography, which comprises the steps of firstly carrying out enzymolysis on alkaline sites of a protein sample into polypeptide, blocking free amino at the N terminal and the side chain of the peptide segments at the level of the peptide segments, then adopting anion exchange chromatography to pre-enrich the polypeptide and reserve stronger SUMO peptide segments, then carrying out SUMO removal on the collected SUMO peptide segments by using SUMO removal enzyme, weakening the reservation of the peptide segments subjected to SUMO removal in the anion exchange chromatography, carrying out anion exchange chromatography on the sample again, collecting the flow components which are not reserved, and realizing secondary enrichment of the SUMO modified peptide segments by separating the SUMO modified substrate peptide segments from other interfering peptide segments. The invention has the advantages of high selectivity and high enrichment efficiency, can simultaneously enrich various types of SUMO modified peptide fragments, and improves the identification coverage of SUMO modified sites.

Description

SUMOylated peptide enrichment method based on de-SUMOylase and SAX

technical field

The present invention relates to a method for enriching SUMOylated peptides, namely a method for enriching SUMOylated peptides based on de-SUMOylase and anion exchange chromatography (SAX), so as to achieve high efficiency and high efficiency of SUMOylated peptides in complex protein samples. Highly selective enrichment.

Background technique

Ubiquitination is one of the common post-translational modifications in organisms, and it is also the first modification that is found to be linked to proteins, and it plays an important role in protein degradation. In the past ten years, scientists have discovered some ubiquitin-like proteins, among which small ubiquitin-like modifiers (SUMO) are the most attention-seeking class.

SUMO plays an important regulatory role in multiple cellular physiological activities, such as maintaining genome stability, regulating cell cycle, regulating cell differentiation and transcription factor activity, and participating in signal transduction. Many SUMO-modified proteins such as transcription factors are in low abundance in cells, and only a small fraction of their protein substrates are modified under normal conditions. When performing mass spectrometry analysis, the concentration range of proteins in complex samples is wide, and high abundance proteins will suppress the mass spectrometry signal of low abundance SUMO-modified proteins. In addition, during the analysis of protein SUMO-modified peptides (sites), a large number of unmodified peptides in SUMO-modified protein enzymatic hydrolysates greatly interfered with SUMO-modified peptides. The above factors seriously affect the identification and quantification efficiency of SUMO-modified proteins and their modification sites.

To improve the identification efficiency of SUMO modifications, various biomarker-based enrichment methods have been developed. Usually, after His-tag marking or mutation of SUMO, Ni-NTA or antibody is used for affinity enrichment of SUMO-modified proteins and polypeptides (Nature Communication 2017, 8, 14109; Nature Structural & Molecular Biology 2017, 24, 325-336; Nature Communication 2015 , 6,7289). Such methods help to improve the identification efficiency of SUMO modifications, but are only applicable to biological samples (such as cells) that can be genetically altered, and cannot be used for samples such as tissues and blood; in addition, changes to the SUMO sequence may affect the properties of SUMO. and function, and therefore cannot accurately reflect the true modification state of the protein in the sample. Several recently published articles have been reported to successfully identify endogenous SUMOylated peptides in human cells and mouse tissues (Nature Communication 2018, 9, 2456; Nature Communication 2017, 8, 1171; Molecular & Cellular Proteomics 2017, 16, 717-727), but still rely on expensive antibodies for affinity enrichment of SUMO-modified polypeptides, and can only enrich for one type of SUMO-modified peptides. Therefore, there is an urgent need to develop a universal enrichment method for endogenous SUMO-modified proteins and modified polypeptides.

In order to overcome the problems of the above methods and establish an efficient and universal enrichment method, we used the SUMOylated peptide segment after the basic site digestion to have the characteristics of polyacidic amino acids, combined with de-SUMOylase and anion. Exchange chromatography achieves efficient removal of non-SUMOylated peptides and improves the selectivity and efficiency of SUMOylated peptide enrichment.

SUMMARY OF THE INVENTION

The present invention develops a method for enriching SUMO modified peptides based on de-SUMOylase and anion exchange chromatography, which has simple and convenient operation steps, high selectivity and high enrichment efficiency.

In order to achieve this purpose, the technical scheme of the present invention is:

1) Block the free amino groups of the N-terminal and side chains in the polypeptide

Dissolve the polypeptide sample produced by the alkaline site of the protein in a buffer with a pH of 6-10, add an amino-active reagent with a final concentration of 10-4000mM, and react for 1-48h, then desalin the solution and freeze-dried , get sample A;

The enzymes used in the alkaline site digestion of proteins are one or more of trypsin, endoproteinase Lys-C, and endoproteinase Arg-C;

pH 6-10 buffers include sodium dihydrogen phosphate, sodium bicarbonate, 4-hydroxyethylpiperazineethanesulfonic acid, triethylenediamine carbonate, 2-(N-morpholine)ethanesulfonic acid, hydrogen phosphate One or more of disodium is configured; the buffer concentration is 10-500mM;

Amino reactive reagents are aldehydes (such as one or more of formaldehyde and acetaldehyde), acid anhydrides (such as one or more of acetic anhydride and propionic anhydride), halides (such as ethyl iodide) One or more of amide, chloroacetamide), guanidylating reagent (such as: one or more of O-methylisourea, 1-H-pyrazole formamidine hydrochloride), Succinylation reagents (such as: one or more of succinimide esters), one or more of Traut's reagents;

When using aldehyde reagents, additionally add sodium cyanoborohydride to a final concentration of 10-600 mM.

2) Use anion exchange chromatography to pre-enrich the SUMOylated peptides retained in the peptides

The obtained sample A was dissolved in phase A and loaded onto an anion exchange chromatographic column for separation, first eluted with a mobile phase containing a molar concentration of salt ≤ 100mM to remove weakly retained enzymolysis peptides, and then using a salt-containing mobile phase The mobile phase with molar concentration >150mM is eluted and the effluent of peptides with strong retention is collected, and the solution is desalted and lyophilized to obtain sample B;

The mobile phase is calculated by volume percentage, phase A: 5-30% organic solvent+1-50mM aqueous solution of alkali; B phase: 5%-30% organic solvent+1-50mM aqueous alkali+200-1000mM salt;

The organic solvent includes one or more of acetonitrile, alcohols, dimethyl sulfoxide, N,N-dimethylformamide, ethers, acetone, pyridine, and chloroform;

The aqueous alkali solution is an ammonium bicarbonate buffered saline solution with a pH of 8-14, a phosphate buffered saline solution with a pH of 8-14, a tris buffered saline solution with a pH of 8-14, ammonia water, sodium carbonate solution, Or one or more of sodium hydroxide solution are configured;

Salts include one or two or more of halogen salts, phosphates, nitrates, carboxylates, carbonates, and sulfates whose cations are one or more of potassium and sodium.

3) De-SUMOylation of the collected SUMOylated peptides with de-SUMOylase

The obtained sample B was dissolved in a buffer with a pH of 4-10, and de-SUMOylase was added to carry out the de-SUMOylation reaction. 72h, the enzymatic hydrolysis temperature is 16-56℃, and the solution C is obtained;

The de-SUMOylase is one or more of SENP1, SENP2, SENP3, SENP5, SENP6, SENP7 and SENP8.

4) Pass the sample through anion exchange chromatography again and collect the unretained fractions

The obtained solution C is loaded on an anion exchange chromatographic column, eluted with phase A of step (2), and the unretained enzymolysis peptides existing in the elution effluent of phase A are collected, which is the depletion after enrichment. SUMOylated substrate peptides.

The beneficial effects of the present invention are:

1. The high reaction efficiency of amino-active reagents for labeling amino groups greatly reduces the false positive identification of SUMO-modified peptides;

2. The strong separation ability of anion exchange chromatography promotes the efficient removal of non-SUMOylated peptides;

3. De-SUMOylase has strong specificity for SUMOylated modified peptides and high enzyme cleavage efficiency;

4. There is no discrimination against different types of SUMOylated peptides to avoid the loss of SUMOylated peptides;

The invention has the advantages of high enrichment selectivity, high enrichment efficiency, simultaneous enrichment of multiple types of SUMOylated peptides, and improved identification coverage of SUMOylated modified sites.

Description of drawings

Figure 1. Enrichment flow of SUMOylated peptides.

Figure 2a) MALDI-TOF mass spectrum of SUMO1-labeled peptide; b) MALDI-TOF mass spectrum of SUMO1-labeled peptide cleaved by de-SUMOylase.

Note: The mass-to-charge ratio of the standard peptide is 3848, and the sequence is ELGMEEEDVIEVYQEQTGG(19)-LLVHMGLLKSEDKVK(9).

Fig. 3 Chromatographic peaks of SUMO1 labeled peptide in anion exchange column.

Figure 4. Chromatographic peaks in anion-exchange chromatography column after SUMO1-labeled peptide was digested by de-SUMOylase.

Detailed ways

Example 1

As shown in Figure 1, the basic site of the protein is first digested, and then the N-terminus of the peptide and the free amino group of the side chain are blocked at the peptide level. The digested SUMO peptide has multiple acidic amino acids. The retention in anion exchange chromatography in an alkaline environment is stronger than that of non-SUMOylated peptides, so anion exchange chromatography is used to pre-enrich the SUMOylated peptides with strong retention, and then the collected SUMOylated peptides are used for SUMOylase is de-SUMOylated, and the retention of de-SUMOylated peptides in anion exchange chromatography is weakened, so that the enrichment of SUMOylated peptides is achieved in the second anion exchange chromatography.

Take the SUMO1 standard peptide with the sequence ELGMEEEDVIEVYQEQTGG(19)-LLVHMGLLKSEDKVK(9) as the sample, dissolve it in 50mM ammonium bicarbonate, and digest it with the de-SUMOylase SENP1, where the amount of enzyme is 1/20 of the sample mass, and the temperature is MALDI-TOF mass spectrometry analysis was performed at 37°C after enzymatic hydrolysis for 12 hours. As shown in Figure 2, two peptides were generated after enzymatic digestion: ELGMEEEDVIEVYQEQTGG (mass-to-charge ratio of 2178), LLVHMGLLKSEDKVK (mass-to-charge ratio of 1710), indicating SUMO The isopeptide bonds of the peptides are efficiently and selectively cleaved.

Example 2

Redissolve 10 μg of SUMO1 standard peptide in phase A, load the sample into a tertiary amine-based ion exchange column (4.6mm i.d×5cm) for isocratic elution, elution gradient (V/V): 0-10min, 20%B; 10-20 min, 80% B, flow rate 1.0 mL/min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM sodium chloride. As shown in Figure 3, after the unretained fraction 10 min before elution, the fraction containing the SUMO1 target peptide after 10 min was collected, desalted, and lyophilized. After redissolving in 0.1% formic acid, mass spectrometry analysis showed that the SUMO1 standard peptide was effectively retained and enriched in anion exchange chromatography.

Example 3

10μg of SUMO1 standard peptide fragment was digested with de-SUMOylase and loaded onto a tertiary amine ion exchange column (4.6mm i.d×5cm) for isocratic elution, elution gradient: 0-10min, 20%B; 10-20 min, 80% B, flow rate 1.0 mL/min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM sodium chloride. As shown in FIG. 4 , the unretained fractions in the first 10 min and the retained fractions after 10 min were collected, demineralized, and lyophilized. Redissolved in 0.1% formic acid for mass spectrometry analysis, the de-SUMOylated peptide LLVHMGLLKSEDKVK after being digested by de-SUMOylase did not retain the efflux in anion exchange, the original SUMO1 standard peptide and the peptide obtained after digestion ELGMEEEDVIEVYQEQTGG was still retained in the anion exchange, indicating that the effective enrichment and identification analysis of SUMOylated peptides was successfully achieved.

Example 4

Taking HeLa cells as the sample, 1000 μg of extracted protein was dissolved in 100 μl of 8M urea in 50 mM ammonium bicarbonate buffer (pH 8), 10 μl of 100 mM dithiothreitol was added, and after denaturation and reduction at 56°C for 1 h, 10 μl of 300 mM iodoacetamide was added to react After 0.5h, another 30μl of 100mM dithiothreitol was added, and after 10min incubation, trypsin digestion was used, where the amount of enzyme was 1/10 of the sample mass, and the temperature was 37°C. After enzymatic hydrolysis for 60min, desalted and lyophilized . The peptides were dissolved in 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer, added with final concentrations of 40 mM formaldehyde and 20 mM sodium cyanoborohydride, respectively, at 37 °C, reacted for 1 h, and loaded onto a tertiary amine ion exchange column (4.6 mm i.d×5cm) for isocratic elution, elution gradient: 0-10min, 25%B; 10-20min, 80%B, flow rate is 1.0mL/min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM sodium chloride. Peptides were not retained for 10 minutes before elution, and the fractions after 10 minutes were collected, desalted, and lyophilized. The peptide fragment was dissolved in 50 mM ammonium bicarbonate, and digested with the de-SUMOase SENP1, where the amount of enzyme was 1/20 of the sample mass, and the temperature was 37 °C. 4.6mm i.d×5cm) for isocratic elution, elution gradient: 0-10min, 20%B; 10-20min, 80%B, flow rate is 1.0mL/min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM sodium chloride. The unretained fractions 10 min before collection were demineralized and lyophilized. That is, the enriched de-SUMOylated peptide sample obtained after being digested by de-SUMOylase.

Example 5

Taking HeLa cells as the sample, 1000 μg of extracted protein was dissolved in 100 μl of 8M urea in 50 mM ammonium bicarbonate buffer (pH 8), 10 μl of 100 mM dithiothreitol was added, and after denaturation and reduction at 56°C for 1 h, 10 μl of 300 mM iodoacetamide was added to react After 0.5 h, another 30 μl of 100 mM dithiothreitol was added, and after 10 min of incubation, endoproteinase Lys-C was used for digestion, where the amount of enzyme was 1/10 of the sample mass, the temperature was 37 °C, and after enzymatic hydrolysis for 60 min, Desalted and lyophilized. The peptides were dissolved in 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer, added with final concentrations of 40 mM formaldehyde and 20 mM sodium cyanoborohydride, respectively, at 37 °C, reacted for 1 h, and loaded onto a tertiary amine ion exchange column (4.6 mm i.d×5cm) for gradient elution, elution gradient: 0-10min, 5-25%B; 10-20min, 25%B, 20-30min, 80%B, the flow rate is 1.0mL/min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM potassium chloride. Peptides that did not remain in the first 20 min of elution were collected after 20 min, desalted, and lyophilized. The peptide fragment was dissolved in 50 mM ammonium bicarbonate, and digested with the de-SUMOase SENP1, where the amount of enzyme was 1/20 of the sample mass, and the temperature was 37 °C. 4.6mmi.d×5cm) for isocratic elution, elution gradient: 0-10min, 20%B; 10-20min, 80%B, flow rate is 1.0mL/min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM sodium chloride. The unretained fractions 10 min before collection were demineralized and lyophilized. That is, the enriched de-SUMOylated peptide sample obtained after being digested by de-SUMOylase.

Example 6

Taking HeLa cells as the sample, 1000 μg of extracted protein was dissolved in 100 μl of 8M urea in 50 mM ammonium bicarbonate buffer (pH 8), 10 μl of 100 mM dithiothreitol was added, and after denaturation and reduction at 56°C for 1 h, 10 μl of 300 mM iodoacetamide was added to react After 0.5 h, another 30 μl of 100 mM dithiothreitol was added, and after 10 min of incubation, endopeptidase Arg-C was used for digestion, where the amount of enzyme was 1/10 of the sample mass, the temperature was 37 °C, and after enzymatic hydrolysis for 60 min, Desalted and lyophilized. The peptide fragment was dissolved in 20mM sodium bicarbonate buffer, added with a final concentration of 10mM succinimidyl acetate, reacted at 37°C for 1h, and loaded onto a tertiary amine-based ion exchange column (4.6mm i.d×5cm) for isocratic Elution, elution gradient: 0-10 min, 25% B; 10-20 min, 80% B, flow rate 1.0 mL/min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM sodium chloride. Peptides were not retained for 10 minutes before elution, and the fractions after 10 minutes were collected, desalted, and lyophilized. The peptide fragment was dissolved in 50 mM ammonium bicarbonate and digested with the de-SUMOase SENP2, where the amount of enzyme was 1/20 of the sample mass, and the temperature was 37 °C. 4.6mm i.d×5cm) for isocratic elution, elution gradient: 0-10min, 20%B; 10-20min, 80%B, flow rate is 1.0mL/min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM potassium nitrate.

The unretained fractions 10 min before collection were demineralized and lyophilized. That is, the enriched de-SUMOylated peptide sample obtained after being digested by de-SUMOylase.

Claims (5)

1. A SUMOylated peptide enrichment method based on de-SUMOylase and anion exchange chromatography SAX, the steps include: 1) Block the free amino groups of the N-terminus and side chain in the polypeptide; the free amino groups of the N-terminus and side chain of the blocked polypeptide, the specific steps are as follows: Dissolve the polypeptide sample produced by the enzyme cleavage at the alkaline site of the protein in a buffer with a pH of 6-10, add an amino-active reagent with a final concentration of 10-4000 mM, and after the reaction for 1-48 h, the solution is desalted. Freeze-drying to obtain sample A; the enzymes used for the alkaline site digestion of the protein are one or more of trypsin, endoproteinase Lys-C, and endopeptidase Arg-C; The active reagent is one or more of aldehydes, acid anhydrides, halides, guanidylation reagents, succinylation reagents, and Traut's reagents; 2) Use anion exchange chromatography SAX to pre-enrich the SUMOylated peptides retained in the peptides; 3) The collected SUMOylated peptides are then de-SUMOylated with de-SUMOylated enzyme; 4) Pass the sample through anion exchange chromatography SAX again, and collect the unretained fractions; Thus, the secondary enrichment of SUMOylated peptides can be achieved. 2. according to the said enrichment method of claim 1, it is characterized in that: In the step 2), the SUMOylated peptides retained in the peptides are pre-enriched by SAX by anion exchange chromatography, and the specific steps are as follows: The obtained sample A was dissolved in phase A and loaded onto an anion exchange chromatographic column for separation, firstly eluted with a mobile phase containing a molar concentration of salt ≤ 100 mM to remove weakly retained enzymolysis peptides, and then using a salt-containing mobile phase The mobile phase with a molar concentration >150 mM was eluted and the effluent of the peptide fragment with strong retention was collected, and the solution was desalted and lyophilized to obtain sample B; The mobile phase is calculated by volume percentage, A phase: 5-30% organic solvent + 1-50 mM alkali in water; B phase: 5%-30% organic solvent + 1-50 mM alkali in water + 200-1000 mM Salt; The organic solvent includes one or more of acetonitrile, alcohols, dimethyl sulfoxide, N,N-dimethylformamide, ethers, acetone, pyridine, and chloroform; The aqueous solution of the alkali is an ammonium bicarbonate buffered saline solution with a pH of 8-14, a phosphate buffered saline solution with a pH of 8-14, a tris buffered saline solution with a pH of 8-14, ammonia water, carbonic acid One or more of sodium solution or sodium hydroxide solution are configured; Described salt comprises one or more in halogen salt, phosphate, nitrate, carboxylate, carbonate, sulfate that cation is one or more in potassium and sodium; In the step 3), the collected SUMOylated peptides are de-SUMOylated with de-SUMOylated enzyme, and the specific steps are as follows: The obtained sample B was dissolved in a buffer with a pH of 4-10, and de-SUMOylase was added to carry out the de-SUMOylation reaction. For 72 h, the enzymatic hydrolysis temperature was 16-56 °C to obtain solution C; In the step 4), the sample is again subjected to anion exchange chromatography SAX, and the unretained fractions are collected. The specific steps are as follows: The obtained solution C was loaded onto an anion exchange chromatographic column, eluted with phase A of step (2), and the unretained enzymolysis peptides existing in the elution effluent of phase A were collected, namely the enriched detoxification. SUMOylated substrate peptides. 3. The method according to claim 2, wherein: Described pH is 6-10 buffer solution including sodium dihydrogen phosphate, sodium bicarbonate, 4-hydroxyethyl piperazine ethanesulfonic acid, triethylenediamine carbonate, 2-(N-morpholine) ethanesulfonic acid , one or two or more of disodium hydrogen phosphate; the buffer concentration is 10-500 mM; The aldehydes of the amino active reagent are one or more of formaldehyde and acetaldehyde, the acid anhydrides are one or more of acetic anhydride and propionic anhydride, and the halide is one or more of iodoacetamide and chloroacetamide. One or more than two, the guanidylating reagent is one or more of O-methylisourea, 1-H-pyrazole carboxamidine hydrochloride, and the succinylating reagent is succinimidyl esters one or more of two; When using aldehyde reagents, additionally add sodium cyanoborohydride to a final concentration of 10-600 mM. 4. according to the method described in claim 2, it is characterized in that: Step 3) The de-SUMOylase is one or more of SENP1, SENP2, SENP3, SENP5, SENP6, SENP7, and SENP8. 5. The application of the method according to claim 1 in the enrichment and identification of SUMOated modified proteins and peptide segments in biological samples; biological samples are one or two of human or animal or plant cells, tissues, and body fluids above.

Images