Application of corn starch in proteomic sample pretreatment

Abstract

The application discloses application of corn starch in proteomics sample pretreatment, and a step of enriching glycopeptides in target glycosylated peptides by using corn starch is as follows: step one, preparing a sample buffer by configuring trifluoroacetic acid, deionized water and acetonitrile according to a certain volume ratio; step two, incubating a solution of corn starch, the sample buffer and the target glycosylated peptides at a predetermined temperature; step three, washing three times with the sample buffer, then adding an elution buffer prepared by configuring trifluoroacetic acid, deionized water and acetonitrile according to a certain volume ratio, eluting at a predetermined temperature for a predetermined time, and centrifugally separating to obtain supernatant; and step four, detecting the supernatant by using MALDI-TOF MS or NanoLC-MS / MS. The corn starch is used for enriching glycopeptides based on hydrophilic interaction liquid chromatography, glycopeptides in a sample are effectively enriched, and direct application of the corn starch avoids complicated synthesis methods, long material synthesis time, high cost and possible pollution caused by chemicals to the environment.

Description

Technical Field

[0001] This invention relates to the field of proteomics analysis technology, and in particular to the application of corn starch in proteomics sample pretreatment. Background Technology

[0002] Post-translational modifications (PTMs) play a crucial role in expanding the complexity of the proteome. Glycosylation, as one of the most important and fundamental PTMs, is widely present in proteins secreted by the human body. Protein glycosylation participates in various essential cellular activities, such as signal transduction, cell proliferation, and apoptosis. Furthermore, the occurrence of some diseases is closely related to abnormal protein glycosylation, such as neurodegenerative diseases, genetic diseases, lung diseases, diabetes, and cancer. Meanwhile, research on COVID-19 has shown that glycosylated proteins of the coronavirus are closely related to receptor binding and cell invasion. Therefore, elucidating information on the degree and location of glycosylation is crucial for timely diagnosis and subsequent targeted therapy. The difference in glycosylation levels between healthy and diseased individuals is relatively small, thus requiring comprehensive analysis of protein glycosylation levels using effective tools. Mass spectrometry (MS) plays a dominant role in protein glycosylation analysis due to its advantages of high throughput, ease of operation, high sensitivity, and immediacy. However, during direct detection of biological samples, the signal of glycopeptides is easily suppressed by interference from other highly abundant non-glycopeptides and impurities. Therefore, selective enrichment and separation of glycopeptides are crucial before MS detection.

[0003] In previous glycoprotein / glycopeptide enrichment strategies, hydrophilic interaction liquid chromatography (HILIC) has been widely used due to the higher hydrophilicity of glycopeptides compared to non-glycopeptides. HILIC offers excellent MS compatibility, does not damage the structure of glycopeptides, and provides selective separation. However, materials for enriching glycopeptides based on HILIC are limited by problems such as high raw material costs, high chemical consumption, environmental unfriendliness, long processing time, and complex material preparation processes.

[0004] Therefore, there is a new need to develop a natural and environmentally friendly hydrophilic interaction liquid chromatography material for the selective separation and enrichment of glycopeptides. To this end, we propose the application of corn starch in proteomics sample pretreatment. Summary of the Invention

[0005] The purpose of this invention is to provide the application of corn starch in proteomics sample pretreatment to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for using corn starch in proteomics sample pretreatment, comprising the following steps for enriching glycopeptides in target glycosylated peptides using corn starch:

[0007] Step 1: Prepare a sample loading buffer by mixing trifluoroacetic acid, deionized water, and acetonitrile in a specific volume ratio.

[0008] Step 2: Incubate the solution containing corn starch, loading buffer, and the target glycosylated peptide at a predetermined temperature for a predetermined time.

[0009] Step 3: Wash three times with loading buffer, then add elution buffer prepared with trifluoroacetic acid, deionized water and acetonitrile in a certain volume ratio, elute at a predetermined temperature for a predetermined time, and centrifuge to obtain supernatant;

[0010] Step 4: Analyze the supernatant obtained in Step 3 using MALDI-TOF MS or NanoLC-MS / MS.

[0011] Preferably, the corn starch is natural corn starch, and the corn starch does not require any other pretreatment processing.

[0012] Preferably, the loading buffer consists of 3% trifluoroacetic acid (v / v), 7% deionized water (v / v), and 90% acetonitrile (v / v).

[0013] Preferably, the elution buffer consists of 0.1% trifluoroacetic acid (v / v), 69.9% deionized water (v / v), and 90% acetonitrile (v / v).

[0014] Preferably, the incubation temperature in step two is 37°C, and the elution temperature in step three is 37°C.

[0015] Preferably, the incubation time in step two is two hours, and the elution time in step three is one hour.

[0016] The technical effects and advantages of this invention are as follows:

[0017] This invention utilizes natural, renewable, and biodegradable corn starch to enrich glycopeptides using hydrophilic interaction liquid chromatography. Due to its carbon-based biomass and porous structure, eggshells without complex pretreatment can effectively enrich glycopeptides in the sample. The direct application of corn starch avoids cumbersome synthesis methods, long material synthesis time, high cost, and potential environmental pollution caused by chemicals. Attached Figure Description

[0018] Figure 1 This is a scanning electron microscope image of corn starch from Embodiment 1 of the present invention;

[0019] Figure 2 This is the mass spectrum of HRP-enriched glycopeptides from corn starch in Example 2 of the present invention;

[0020] Figure 3This is the mass spectrum of the detection limit (0.1 fmol / μL) of corn starch-enriched glycopeptides in Example 2 of the present invention;

[0021] Figure 4 Mass spectrum of glycopeptides in corn starch-enriched mixed protein (BSA:HRP=2000:1) in Example 3 of the present invention;

[0022] Figure 5 This is the mass spectrum of the corn starch-enriched standard glycopeptides in Example 4 of the present invention;

[0023] Figure 6 This is a Venn diagram of glycopeptide fragments from the serum exosomes of three healthy individuals and three colorectal cancer patients enriched with corn starch in Example 5 of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1

[0026] Scanning electron microscope images of the obtained corn starch are as follows: Figure 1 As shown.

[0027] Example 2

[0028] Application of corn starch in the enrichment of glycopeptides from HRP:

[0029] (1) Sample preparation: Horseradish peroxidase (HRP) was enzymatically hydrolyzed in 25 mmol / L NH4HCO3 solution at 37°C for 16 hours;

[0030] (2) Enrichment: 0.5 mg corn starch powder was dispersed into a centrifuge tube containing 100 μL of 3% trifluoroacetic acid, 7% deionized water and 90% acetonitrile, and 2 μL of the sample prepared in step (1) was added. The mixture was enriched at 37°C for 2 hours. The mixture was washed thoroughly with a buffer solution of 3% trifluoroacetic acid, 7% deionized water and 90% acetonitrile and centrifuged 3 times. 10 μL of an elution buffer of 0.1% trifluoroacetic acid, 30% acetonitrile and 69.9% deionized water was added. The mixture was eluted at 37°C for 1 hour and the supernatant was obtained by centrifugation.

[0031] (3) Mass spectrometry analysis: Take 1 μL of the supernatant obtained in step (2) and spot it onto the target. Use DHB as the matrix for mass spectrometry analysis. The mass spectrum is shown in the figure. Figure 3 As shown, Figure 4 It is the detection limit for glycopeptides enriched in HRP by corn starch (0.1 fmol / μL).

[0032] From Example 2 and Figure 3 The HRP hydrolysate was used to evaluate the ability of corn starch to enrich glycopeptides. It was found that glycopeptides are captured through the hydrophilic interaction between biomass and glycopeptides. When detected directly without enrichment, non-glycopeptide signals dominated the spectrum, greatly suppressing glycopeptide signals. However, after using corn starch to specifically enrich glycopeptides, the signals of glycopeptides increased significantly, with 21 glycopeptide signals detected, and these glycopeptide signals dominated the spectrum. There were almost no non-glycopeptide signals in the mass spectrum, indicating that corn starch has an excellent ability to enrich glycopeptides.

[0033] From Example 2 and Figure 4 It can be seen that the ability of corn starch to enrich glycopeptides was evaluated using a low concentration of HRP hydrolysate diluted to 0.1 fmol / μL. The enrichment of glycopeptides at low abundance was achieved by relying on the excellent hydrophilic interaction between starch and glycopeptides. Figure 4 As can be seen, corn starch can still capture 3 glycopeptides in a low concentration of HRP hydrolysate of 0.1 fmol / μL, indicating that corn starch has a low detection limit for glycopeptides.

[0034] Example 3

[0035] Application of corn starch in enriching glycopeptides in mixed proteins:

[0036] (1) Sample preparation: Horseradish peroxidase (HRP) was enzymatically hydrolyzed in 25mM NH4HCO3 solution at 37℃ for 16 hours; Bovine serum albumin (BSA) and horseradish peroxidase (HRP) hydrolysate were added at a molar ratio of 2000:1 to a centrifuge tube containing 3% trifluoroacetic acid, 7% deionized water and 90% acetonitrile.

[0037] (2) Enrichment: 0.5 mg of the material obtained in Example 1 was dispersed into 100 μL of centrifuge tube containing 3% (v / v) trifluoroacetic acid, 7% (v / v) deionized water and 90% (v / v) acetonitrile of the glycopeptide in step (1), and enriched at 37°C for 2 hours; washed thoroughly with 3% (v / v) trifluoroacetic acid, 7% (v / v) deionized water and 90% (v / v) acetonitrile buffer and centrifuged 3 times; added 0.1% (v / v) trifluoroacetic acid, 30% (v / v) acetonitrile and 69.9% (v / v) deionized water elution buffer, eluted at 37°C for 1 hour, and centrifuged to obtain the supernatant;

[0038] (3) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (2), spot the target, and perform mass spectrometry analysis using DHB as the matrix. Figure 5This is a mass spectrum of glycopeptides in corn starch-enriched mixed proteins (BSA:HRP=2000:1).

[0039] As shown in Example 3, using BSA as an interfering protein, the selectivity of corn starch for HRP was studied by changing the mass ratio of HRP hydrolysate to BSA. Without enrichment and direct detection, the obtained mass spectrum showed no detected glycopeptides. After using corn starch to specifically enrich glycopeptides, even with a mass ratio of HRP hydrolysate to BSA of 1:2000, corn starch still enriched 6 glycopeptides, indicating that corn starch has excellent selectivity for HRP and can capture glycopeptides from semi-complex samples.

[0040] Example 4

[0041] Analysis of glycopeptide loading in HRP-enriched corn starch;

[0042] (1) Weigh out 25μg, 50μg, 75μg, 100μg and 150μg of corn starch powder;

[0043] (2) Enrichment: Different amounts of corn starch powder from step (1) were dispersed into centrifuge tubes containing 100 μL of glycopeptide in 3% (v / v) trifluoroacetic acid, 7% (v / v) deionized water, and 90% (v / v) acetonitrile. 0.01 mg HRP was added to each tube, and the mixture was enriched at 37°C for 2 hours. The tubes were washed thoroughly with 3% (v / v) trifluoroacetic acid, 7% (v / v) deionized water, and 90% (v / v) acetonitrile buffer and centrifuged 3 times. 10 μL of 0.1% (v / v) trifluoroacetic acid, 30% (v / v) acetonitrile, and 69.9% (v / v) deionized water eluent was added to each tube, and the mixture was eluted at 37°C for 1 hour. The supernatant was obtained by centrifugation.

[0044] (3) Mass spectrometry analysis: Take 1 μL of the five eluents obtained in step (2) and spot them onto the target. Use DHB as the matrix for mass spectrometry analysis. The mass spectrum is shown below. Figure 6 As shown, the calculated loading capacity of the material for glycopeptides is 133 mg / g.

[0045] From Example 4 and Figure 6 It can be seen that by enriching glycopeptides from the same amount of HRP (0.01 mg) enzymatic hydrolysate solution using different amounts (25 μg-125 μg) of corn starch powder, and by evaluating the intensity of three prominent glycopeptide peaks through three parallel tests, the loading capacity of corn starch for glycopeptides was studied. Figure 6As shown, the peak intensities of the three glycopeptides (m / z = 3672, 4838, 4983) increased with increasing material quantity. The intensities of all three glycopeptide peaks reached their peak values ​​when the material quantity was 75 μg. Although the material quantity increased, the peak intensities of the glycopeptides did not change significantly. This indicates that 0.01 mg HRP was enriched to saturation by 75 μg of material. By dividing the amount of HRP (0.01 mg) by the amount of material at saturation (75 μg), the loading capacity of corn starch for glycopeptides was calculated to be approximately 133 mg / g.

[0046] Example 5

[0047] Application of corn starch in enriching glycopeptides in human serum;

[0048] (1) Sample preparation: The obtained human serum was first reduced and alkylated with dithiothreitol and iodoacetamide, and then enzymatically digested at 37°C for 16 hours;

[0049] (2) Enrichment: 0.5 mg corn starch powder was dispersed into 100 μL centrifuge tube containing 3% (v / v) trifluoroacetic acid, 7% (v / v) deionized water and 90% (v / v) acetonitrile of the glycopeptide from step (1), and enriched at 37°C for 2 hours; washed thoroughly with 3% (v / v) trifluoroacetic acid, 7% (v / v) deionized water and 90% (v / v) acetonitrile buffer and centrifuged 3 times; added 10 μL of 0.1% (v / v) trifluoroacetic acid, 30% (v / v) acetonitrile and 69.9% (v / v) deionized water elution buffer, eluted at 37°C for 1 hour, and centrifuged to obtain the supernatant;

[0050] (3) Mass spectrometry analysis: The eluent obtained in step (2) was collected, deglycosylated at 37°C for 16 hours, desalted and lyophilized, and then analyzed by Nano-LC-MS / MS. The Venn diagram of the obtained data is shown below. Figure 6 As shown.

[0051] From Example 5 and Figure 6 It can be seen that using serum exosomal trypsin digests from three healthy individuals and serum exosomal trypsin digests from three colorectal cancer patients as typical complex biological samples for enrichment experiments, Figure 6 To investigate the differences in glycopeptides, glycosylation sites, and glycoprotein quantities from serum trypsin digests of three healthy individuals and serum exosomal trypsin digests of three colorectal cancer patients, a Venn diagram was created to enrich and identify these glycopeptides, glycosylation sites, and glycoprotein quantities. Figure 6The results showed that 187 N-glycopeptides and 134 glycosylation sites belonging to 78 glycoproteins were identified from the serum exosomal trypsin digests of three healthy individuals, while 114 N-glycopeptides and 124 glycosylation sites belonging to 191 glycoproteins were identified from the serum exosomal trypsin digests of three colorectal cancer patients. These results indicate that corn starch has a good potential for enriching glycopeptides and is expected to be used as a natural and green material for the separation of glycopeptides from complex biological samples, showing promising application prospects in glycoproteomics.

[0052] Examples 1-5 demonstrate the enrichment of glycopeptides using natural, renewable, and biodegradable corn starch based on hydrophilic interaction liquid chromatography. Corn starch without complex pretreatment can effectively enrich glycopeptides in samples. The direct application of starch avoids cumbersome synthesis methods, long material synthesis time, high cost, and potential environmental pollution from chemicals. It exhibits extremely high specificity and selectivity for glycopeptides, indicating its promising prospects in proteomics analysis research.

[0053] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of corn starch in proteomics sample pretreatment, characterized in that, The steps for enriching glycopeptides in target glycosylated peptides using corn starch are as follows: Step 1: Prepare a sample loading buffer by mixing trifluoroacetic acid, deionized water, and acetonitrile in a specific volume ratio. Step 2: Incubate the solution containing corn starch, loading buffer, and the target glycosylated peptide at a predetermined temperature for a predetermined time. Step 3: Wash three times with loading buffer, then add elution buffer prepared with trifluoroacetic acid, deionized water and acetonitrile in a certain volume ratio, elute at a predetermined temperature for a predetermined time, and centrifuge to obtain supernatant; Step 4: Analyze the supernatant obtained in Step 3 using MALDI-TOF MS or NanoLC-MS / MS.

2. The application of corn starch in proteomics sample pretreatment according to claim 1, characterized in that, The corn starch is natural corn starch and requires no other pretreatment.

3. The application of corn starch in proteomics sample pretreatment according to claim 1, characterized in that, The loading buffer consists of 3% trifluoroacetic acid (v / v), 7% deionized water (v / v), and 90% acetonitrile (v / v).

4. The application of corn starch in proteomics sample pretreatment according to claim 1, characterized in that, The elution buffer consists of 0.1% trifluoroacetic acid (v / v), 69.9% deionized water (v / v), and 90% acetonitrile (v / v).

5. The application of corn starch in proteomics sample pretreatment according to claim 1, characterized in that, The incubation temperature in step two is 37°C, and the elution temperature in step three is 37°C.

6. The application of corn starch in proteomics sample pretreatment according to claim 1, characterized in that, The incubation time in step two is two hours, and the elution time in step three is one hour.

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