Modified ultrafiltration membrane, preparation method thereof and method for preparing protein sample for mass spectrometry by adopting modified ultrafiltration membrane

Abstract

The invention discloses a modified ultrafiltration membrane, a preparation method thereof and a method for preparing a protein sample for mass spectrometry by adopting the modified ultrafiltration membrane, and belongs to the technical field of protein sample preparation. The surface of the ultrafiltration membrane is subjected to graft modification, so that the hydrophilicity of the surface of the membrane is effectively enhanced, that is, protein adsorption and membrane pollution are reduced by reducing the hydrophobicity of the surface of the membrane and increasing the hydrophilicity. According to the invention, amphoteric small molecules are connected to cellulose molecules of the ultrafiltration membrane, so that hydrophilic ends face outwards to mutually form hydrogen bonds to improve the hydrophilicity. Specifically, according to the method, the membrane is firstly subjected to alkaline activation, then amphoteric small molecules are inoculated, and the connection efficiency is improved. When the modified material is used for preparing a protein sample, the adsorption effect of the membrane on the protein sample can be reduced, the number of identified proteins and peptide fragments is finally increased, protein loss is reduced in the sample pretreatment step, and identification of more proteins and peptide fragments is facilitated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein sample preparation, and particularly relates to a modified ultrafiltration membrane, a preparation method thereof, and a preparation method for preparing a protein sample for mass spectrometry. BACKGROUND

[0002] Prior to 2009, the most commonly used method for preparing a protein sample for mass spectrometry analysis included in-gel digestion and in-solution digestion. The in-gel digestion method separates proteins of different molecular weights by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then the gel is decolorized, cut into small pieces, and subjected to reduction alkylation of disulfide bonds and trypsin digestion to obtain peptide segments. Finally, the peptide segment sample is desalted and subjected to mass spectrometry detection. This method uses sodium dodecyl sulfate (SDS), a highly efficient anionic surfactant, to dissolve most proteins. However, this method is complex, costly, and has high protein loss, and the in-gel digestion efficiency is low and the missed cleavage rate is high. The in-solution digestion method directly digests proteins in solution, and acetone is used to precipitate proteins to remove surfactants such as SDS that are not conducive to mass spectrometry detection. This method has high digestion efficiency and low missed cleavage rate, but it takes a long time, and the use of high-concentration urea to resolubilize the precipitated proteins can cause protein loss and introduce polymers to interfere with mass spectrometry detection.

[0003] In 2009, Wisniewski et al. first proposed a new proteomics pretreatment method, filter-aided sample preparation (FASP), which is a series of reduction alkylation and protein digestion in a regenerated cellulose membrane in an ultrafiltration centrifuge tube. The method uses an ultrafiltration membrane to retain large protein molecules and remove small molecules such as inorganic salts and surfactants in the digestion solution. Finally, the protein is digested using an 8 mol / L urea buffer solution replaced with NH4HCO3, and the digested peptides are collected by centrifugation. This method is time-saving, has little sample contamination, avoids the influence of incompletely digested proteins and excess trypsin on mass spectrometry identification, and also avoids the shortcomings of in-solution digestion and in-gel digestion.

[0004] However, the FASP method also has limitations. The adsorption of proteins to the ultrafiltration tube itself can reduce the recovery rate of peptides. The cause of protein adsorption is the low protein pass rate caused by the hydrophobicity of the ultrafiltration membrane surface, and the increase in the amount of protein retained by the ultrafiltration membrane can increase the filtration resistance of the membrane, reduce the flux, and decrease the separation efficiency. SUMMARY

[0005] In order to overcome the above-mentioned defects of the prior art, the purpose of the present application is to provide a modified ultrafiltration membrane, a preparation method thereof and a preparation method of a protein sample for mass spectrometry analysis, so as to solve the technical problem of low protein peptide recovery rate of the FASP method.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: The present application discloses a preparation method of a modified ultrafiltration membrane, comprising: The regenerated cellulose membrane is soaked and activated in an alkaline solution, the membrane material after the activation treatment is washed, then the membrane material is continuously soaked in a buffer solution containing an amphoteric small molecule, and the surface of the membrane material is grafted and modified under oscillation reaction at 40-55 DEG C for 8-16 hours, and the modified ultrafiltration membrane is prepared after drying.

[0007] Preferably, the regenerated cellulose membrane is cleaned before use, comprising: the regenerated ultrafiltration membrane is first soaked in isopropyl alcohol for 45-60 min, and then washed with water.

[0008] Preferably, the alkaline solution adopts a NaOH solution with a mass concentration of 4%-8%.

[0009] Further preferably, a NaOH solution with a mass concentration of 6% is adopted.

[0010] Preferably, the soaking and activation treatment time is 1-2 h.

[0011] Preferably, the amphoteric small molecule is 1 mol / L glycine.

[0012] The present application discloses a modified ultrafiltration membrane prepared by the above-mentioned preparation method of a modified ultrafiltration membrane, and the adsorption amount of the modified ultrafiltration membrane to protein is reduced.

[0013] The present application discloses an ultrafiltration centrifuge tube prepared from the above-mentioned modified ultrafiltration membrane.

[0014] The present application further discloses a low-adsorption method for preparing a protein sample for mass spectrometry analysis, wherein the protein sample to be analyzed is lysed and incubated, and then transferred to an ultrafiltration centrifuge tube containing the above-mentioned modified ultrafiltration membrane for treatment.

[0015] Specifically, the protein sample to be treated is subjected to lysis treatment: when the cell density in a 6-well plate is close to 80%-90%, the cells are collected, a lysis solution is added, and after mixing by blowing, the cells are lysed for 10 min, and then subjected to high-speed centrifugal treatment for 5 min, and the supernatant is taken and the protein concentration is measured by BCA method.

[0016] Incubation treatment: take the above protein lysate, add UA buffer (8 mol / L urea, 0.1 mol / L Tris-HCl, pH 8.5) and 1 mol / L DTT, incubate at 37°C for 1-2.5 h, transfer to the above modified 10 kD ultrafiltration centrifuge tube, high-speed centrifugal treatment for 20 min, add UA buffer again and repeat centrifugation and discard the effluent in the lower collection tube. Then add UA solution (containing 50 mmol / L IAA) to the upper ultrafiltration tube, incubate in the dark for 40 min, centrifuge and discard the effluent, repeat the addition of UA solution and centrifugal washing once. Finally, add 50 mmol / L NH4HCO3 solution and centrifugal wash twice, discard the effluent and old collection tube, replace the new collection tube, add 50 mmol / L NH4HCO3 solution containing trypsin (trypsin: protein, 1:25-1:20, w / w) to the upper ultrafiltration tube, incubate at 37°C overnight, collect the peptide solution in the lower collection tube after centrifugation, add 50 mmol / L NH4HCO3 to the ultrafiltration tube and centrifugal wash once, and combine the peptide solutions in the collection tubes after the second centrifugation. Desalt the column to remove salt, and dissolve the peptide sample in 20 μL of 0.1% formic acid for mass spectrometry analysis.

[0017] Preferably, the method is used to retain proteins with a molecular weight of 10 kD or more in the sample.

[0018] Further preferably, the optimal protein column load is 20 µg.

[0019] Alternatively, a method for preparing a protein sample for mass spectrometry analysis with low adsorption is an improved FASP sample preparation method. By grafting modification of the ultrafiltration membrane surface in the ultrafiltration centrifuge tube, the hydrophilicity of the membrane surface is enhanced, and the protein adsorption and membrane pollution are reduced. The method comprises the following steps: 1) Preparation of modified ultrafiltration membrane: soak the regenerated cellulose membrane in an alkaline solution for activation treatment, wash the activated membrane material, then continue to soak in a buffer containing small amphoteric molecules, and oscillate at 40-55°C for 8-16 hours for graft modification of the membrane material surface, and dry to obtain a modified ultrafiltration membrane; 2) Protein acquisition and extraction: culture cells, add lysate, mix by blowing, centrifuge to obtain the supernatant, and obtain the protein lysate; 3) Preparation of protein sample: Take the protein lysate, add buffer for incubation, transfer to the 10kD ultrafiltration centrifuge tube (modified ultrafiltration membrane) modified above, centrifuge, add buffer and repeat centrifugation, and discard the effluent in the lower collection tube. Then add buffer to the upper ultrafiltration tube, incubate in the dark, centrifuge and discard the effluent, repeat the addition of buffer and centrifugation for 1 time. Finally, centrifugal washing is performed for 2 times, the effluent and the old collection tube are discarded, a new collection tube is replaced, a solution containing trypsin is added to the upper ultrafiltration tube, incubated at 37 DEG C overnight, and the peptide solution in the lower collection tube is collected after centrifugation. The peptide solution in the two centrifugation collection tubes is combined after washing the ultrafiltration tube with a washing solution for 1 time. After desalting with a desalting column, the peptide sample is dissolved and subjected to mass spectrometry analysis.

[0020] Compared with the prior art, the present application has the following beneficial effects: The present application effectively enhances the hydrophilicity of the membrane surface by grafting modification of the ultrafiltration membrane surface, that is, by reducing the hydrophobicity of the membrane surface to increase the hydrophilicity to reduce protein adsorption and membrane fouling. The present application connects small amphoteric molecules to the cellulose molecules of the ultrafiltration membrane, so that the hydrophilic ends face outward and form hydrogen bonds with each other to improve the hydrophilicity. Specifically, the method first activates the membrane in an alkaline environment, and then connects small amphoteric molecules to improve the connection efficiency.

[0021] The use of the modified material of the present application for protein sample preparation can reduce the adsorption of the membrane to the protein sample, ultimately increase the number of identified proteins and peptides, reduce protein loss in the sample pretreatment step, and help identify more proteins and peptides. This method is expected to provide help in the fields of screening of disease-related biomarkers, drug efficacy mechanism, effect target identification, and diagnosis, monitoring and treatment of diseases.

[0022] Through experimental verification, the ultrafiltration material of the present application successfully grafts small amphoteric molecules to the regenerated cellulose membrane, improves the hydrophilic performance, and reduces the protein adsorption rate. The use of the modified material for protein sample preparation is compared with the preparation of protein sample using the original material without modification (classic FASP method). The results show that, for example, after extracting proteins using NP40 lysate, the improved FASP method of the present application identifies about 7.54% more proteins and about 29.58% more peptides than the traditional FASP method; for another example, after extracting proteins using SDS lysate, the improved FASP method of the present application identifies about 7.20% more proteins and about 37.38% more peptides than the traditional FASP method; for another example, after extracting proteins using TritonX-100, the improved FASP method of the present application identifies about 8.74% more proteins and about 40.13% more peptides than the traditional FASP method. At the same time, it is determined that the optimal protein column load of the improved FASP method of the present application is 20 µg.

[0023] Furthermore, the present invention selects glycine with small steric hindrance to connect to the regenerated cellulose membrane to improve the hydrophilicity of the membrane. However, glycine is not easy to connect and the connection efficiency is low.

[0024] Furthermore, the commercial regenerated cellulose (RC) membrane in the ultrafiltration centrifuge tube was pretreated to remove additives such as glycerol, preservatives and wetting agents that may be present on its surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the structural formula of cellulose molecule; Figure 2 It is the modification principle of RC ultrafiltration membrane; Figure 3 Electron microscopy (a), (b) and Raman spectroscopy (c), (d) characteristics of the membrane before and after modification; Figure 4 The full XPS spectra of RC membranes before and after modification; Figure 5 The water contact angle change diagram of the RC membrane surface before and after modification; Figure 6 The mass spectrometry identification results of three sample pretreatment methods in different lysates are shown; Figure 7 The abundance distribution of peptides detected by mass spectrometry for three sample pretreatment methods in different lysates; Figure 8 Identify the isoelectric point distribution of peptides for three sample pretreatment methods in different lysates; Figure 9 Identify protein molecular weight distribution for three sample preparation methods; Figure 10 The protein and peptide identification results are shown for different protein treatment amounts. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0027] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, unless the context clearly indicates otherwise. It will be further understood that the use of relational terms such as first and second, and the like are used solely to distinguish one from another entity without necessarily implying a relationship or order. It is to be understood that where the application is indicated to include "a" or "an" or "the" or one of something the application also contemplates "one or more" or "at least one", but not excluding additional or other integers.

[0028] The application will be further described in detail by the following embodiments with reference to the accompanying drawings: The application discloses an improved FASP sample preparation method, which reduces the adsorption of proteins on the surface of the ultrafiltration membrane in the ultrafiltration centrifuge tube by grafting modification, so that the preparation effect of protein samples for mass spectrometry detection is significantly improved.

[0029] I. Preparation method The ultrafiltration material in the ultrafiltration centrifuge tube is regenerated cellulose (RC), which can intercept compounds with different molecular weights according to different specifications, and is mainly used to intercept various proteins with a molecular weight of 10 kD or more in the sample in the application, and to reduce, alkylate and trypsin enzymolysis into peptide segments, so as to meet the requirements of subsequent proteomics mass spectrometry analysis.

[0030] The core component of the regenerated cellulose (RC) ultrafiltration membrane is a natural linear polysaccharide macromolecule formed by connecting D-glucosan through 1,4 β-type glycosidic bonds, and its chemical structure can be represented as (C6H 10 O5)n, wherein n represents the degree of polymerization. The structural formula of cellulose molecules is shown in Figure 1 .

[0031] Regenerated cellulose ultrafiltration membrane can reduce protein adsorption by reducing membrane surface hydrophobicity and increasing hydrophilicity. One of the effective methods of membrane modification is to introduce small amphoteric molecules into the cellulose molecules, so that the hydrophilic end faces outward and forms hydrogen bonds to improve hydrophilicity. The present invention selects glycine with small steric hindrance to connect regenerated cellulose membrane to improve the hydrophilicity of the membrane. Previous experiments found that when glycine is directly connected, it is not easy to connect and the connection efficiency is low. The present invention activates the membrane in alkaline, then connects glycine to improve the connection efficiency. At the same time, in order to improve the experimental effect, the commercial regenerated cellulose membrane (RC) in the ultrafiltration centrifuge tube needs to be pretreated to remove the additives such as glycerol, preservatives and wetting agents that may exist on its surface. The modification principle diagram of regenerated cellulose membrane (RC) is shown in Figure 2

[0032] Specifically, the preparation method comprises the following steps: Pretreatment of the original membrane: the RC membrane is soaked in isopropyl alcohol (IPA) for 45-60 min; the RC membrane is washed with ultrapure water to ensure that the surface is clean and free of impurities.

[0033] Membrane activation: the clean RC membrane is soaked in a 6% NaOH solution for 1 h.

[0034] Membrane modification: the activated membrane is taken out and washed clean, then it is soaked in a 1 mol / L glycine (0.1 mol / L Tris-HCl) solution, and oscillated at 45°C for 12 h. After drying, it is ready for use.

[0035] Protein acquisition and extraction: when the cell density of human cervical cancer HeLa cells cultured in a 6-well plate reaches 80%-90%, the cells are collected, 200 μL of lysis solution is added, and after mixing by blowing, the cells are lysed for 10 min. Centrifuged at 11 200 r / min for 5 min, take the supernatant, measure the protein concentration by BCA method.

[0036] ​Preparation method of protein sample: take 20 μg protein lysate, add 200 mL UA buffer (8 mol / L urea, 0.1 mol / L Tris-HCl, pH 8.5) and 2.3 μL 1 mol / L DTT, incubate at 37 ℃ for 1-2.5 h, transfer to the 10 kD ultrafiltration centrifuge tube modified above, centrifuge at 11 200 r / min for 20 min, add 200 μL UA and repeat centrifugation and discard the effluent in the lower collection tube. Then add 100 μL UA solution (containing 50 mmol / L IAA) to the upper ultrafiltration tube, incubate in the dark for 40 min, centrifuge and discard the effluent, repeat the addition of 200 μL UA and centrifugal washing once. Finally, add 200 μL 50 mmol / L NH4HCO3 solution and centrifugal wash twice, discard the effluent and the old collection tube, replace the new collection tube, add 100-200 μL 50 mmol / L NH4HCO3 solution containing trypsin (trypsin: protein, 1:25-1:20, w / w) to the upper ultrafiltration tube, incubate at 37 ℃ overnight, collect the peptide solution in the lower collection tube after centrifugation, add 200 μL 50 mmol / L NH4HCO3 to the ultrafiltration tube and centrifugal wash once, and combine the peptide solutions in the collection tubes after the second centrifugation. After desalting column desalting, dissolve the peptide sample in 20 μL 0.1% formic acid for mass spectrometry analysis.

[0037] II. Characterization and functional verification 1. Characterization Chemical characterization detection was performed on the modified ultrafiltration membrane to verify that glycine was successfully connected to the surface of the ultrafiltration membrane. Laser confocal Raman spectroscopy: The characteristic functional groups on the surface of RC ultrafiltration membrane before and after modification of glycine were tested by laser Raman spectrometer, and the grafting of the surface of the ultrafiltration membrane was verified. The laser with an excitation wavelength of 532 nm and a power of 100 MW was selected, and the Raman spectrum acquisition displacement range was set to 3200-100 cm -1 .

[0038] Field emission scanning electron microscope: After vacuum sputtering treatment by ion sputtering instrument, the sample has conductivity, and the field emission scanning electron microscope is used to characterize the microstructure of the surface of RC ultrafiltration membrane before and after modification of glycine, and to judge the influence of modification treatment on the pore structure of the membrane.

[0039] As shown in Figure 3 , no obvious structural change was found on the surface of the modified RC ultrafiltration membrane, and the pore size of the RC membrane did not change significantly. The Raman spectrum added C-N nitrogen-containing functional groups at 1033 cm -1 and -COO- oxygen-containing functional groups at 1413 cm -1 .

[0040] X-ray photoelectron spectroscopy (XPS): The XPS test was used to explore the changes of the surface chemical composition of the RC ultrafiltration membrane before and after glycine modification. The XPS full spectrum of the unmodified membrane and the glycine modified membrane is shown in Figure 4 As shown in the figure, the two RC membranes mainly contain the skeleton elements C and O. At the same time, in the XPS full spectrum peak graph of the glycine modified membrane, in addition to the main C and O elements, there is also a small amount of N element, which is introduced by the NH2 group of glycine molecules, indicating that the glycine molecules are connected to the surface of the RC membrane.

[0041] 2. Effect verification of the modified ultrafiltration membrane: 2.1 Detection and verification of hydrophilic performance The hydrophilic performance of the ultrafiltration membrane can be directly evaluated by measuring the size of the water contact angle on the membrane surface. The contact angle measuring instrument used in the present application has a measurement range of 0-180° and a resolution of 0.01°. The image is captured instantaneously, and the contact angle of the water droplet with the membrane surface is recorded. As shown in Figure 5 It can be seen that the average contact angle of the unmodified membrane surface is 36.8°, and the average contact angle after glycine modification is reduced to 4.8°. Combined with the previous conclusion, it is confirmed that the change of the chemical composition and structure of the membrane surface after the introduction of glycine enhances the hydrophilicity of the membrane surface, resulting in a decrease in the contact angle.

[0042] 2.2 Verification of the protein adsorption effect of the modified ultrafiltration membrane Protein adsorption rate calculation: The adsorption effect of bovine serum albumin (BSA) was used to measure the protein adsorption amount of the ultrafiltration membrane. Prepare a 1 g / L BSA solution, immerse the RC original membrane and the modified RC membrane (surface area of 1 cm x 1.5 cm) in the 1 g / L BSA solution for 24 h, centrifuge at 11200 r / min for 15 min, collect the effluent, measure the BSA protein content by bicinchoninic acid (BCA) method, and calculate the adsorption amount on the membrane surface according to the following formula.

[0043]

[0044] In the formula, q is the protein adsorption amount; C 0 is the BSA concentration before adsorption; C 1 is the BSA concentration after adsorption; V is the volume of the BSA solution; m is the membrane surface area. The protein adsorption amount on the surface of the unmodified membrane and the glycine modified RC membrane was tested, and the standard protein adsorption amount of BSA was 4.6 μg / cm 2 and 2.8 μg / cm 2 , which indicates that the protein adsorption amount on the surface of the modified membrane is reduced, which means that the anti-protein adhesion performance of the membrane surface is improved.

[0045] Finally, the human cervical cancer cell (HeLa) protein was used as the application object, and the protein sample preparation effect of the method was verified by mass spectrometry detection: the modified RC ultrafiltration membrane was used for protein FASP sample pretreatment (improved FASP), and compared with the classical FASP method and the solution in-solution digestion sample preparation method. At the same time, the influence of the protein extracted by three different surfactant components (NP40 lysis buffer, SDS lysis buffer and TritonX-100 lysis buffer) on the subsequent sample preparation effect was investigated.

[0046] In the three methods, the HeLa protein treatment amount was 20 μg, and three repeated experiments were carried out, and the mass spectrometry injection amount was 2 μL. After three times of mass spectrometry DDA data acquisition, the original data was searched and identified by Proteome Discoverer 2.3 software, and the identification results of the final protein and peptide segment of the three sample preparation methods are shown in Figure 6 It can be seen that after the protein is extracted by the NP40 lysis buffer, the number of proteins identified by the improved FASP method is increased by about 7.54%, and the number of peptides is increased by about 29.58%, as shown in Figure 6 (a). After the protein is extracted by the SDS lysis buffer, the number of proteins identified by the improved FASP method is increased by about 7.20%, and the number of peptides is increased by about 37.38%, as shown in Figure 6 (b). After the protein is extracted by TritonX-100, the number of proteins identified by the improved FASP method is increased by about 8.74%, and the number of peptides is increased by about 40.13%, as shown in Figure 6 (c).

[0047] The peptide segment abundance distribution detected by mass spectrometry under different sample pretreatment methods is shown in Figure 7 It can be seen that after the protein is extracted by the three different lysis buffers, compared with the classical FASP method, the number of peptide segments with abundance greater than 10 4 identified by the improved FASP method is obviously increased, and the number of peptide segments with abundance less than 10 4 is obviously reduced. The improved FASP method can obtain more peptide segments, thereby enhancing the low-abundance peptide segment signal response. In addition, compared with the in-solution digestion sample preparation method, the improved FASP method can detect more peptide segments with abundance greater than 10 6 , and has better reproducibility. The number of proteins and peptide segments identified by the improved FASP method is obviously increased compared with the classical FASP method, and is equivalent to the in-solution digestion sample preparation method.

[0048] Further, the isoelectric point distribution of the peptide segments identified by the three sample pretreatment methods in different lysis buffers and the molecular weight distribution of the identified proteins are shown in Figure 8and Figure 9 It can be seen that the modified FASP method identifies more proteins of each isoelectric point and molecular weight distribution than the classical FASP method, indicating that the method can stably improve the number of identified proteins.

[0049] 2.3 Maximum column load of improved FASP method In the process of sample preparation by FASP method, the optimization of protein column load is crucial for improving the sensitivity and accuracy of protein analysis. Under the condition of keeping the LC-MS injection volume constant at 2 μL, different protein column loads were investigated. The same Western and IP lysis buffer (lysis component 1% Triton X-100) was selected to extract proteins, and 5, 10, 20, 40 and 60 μg of HeLa cell proteins were treated in ultrafiltration tubes respectively. To ensure the reliability and stability of the experimental results, each column load was repeated 3 times. The final sample size of different initial protein treatment amounts was between 0.3 and 2.5 μg to ensure that it did not exceed the maximum column load of the chromatographic analysis column. The protein and peptide identification results of different protein treatment amounts are shown in Figure 10 The optimal column load of the modified RC membrane treated protein is 20 μg (0.5 mL, 10kD, MWCO (Molecular Weight Cut-Off)), which is sufficient for subsequent mass spectrometric analysis.

[0050] In summary, the present application discloses an improved FASP protein sample preparation method for mass spectrometric detection. The improved FASP protein pretreatment method is used in combination with ultrafast high-resolution mass spectrometry to detect LC-MS / MS of human cervical cancer cells (HeLa). The differences in the number of proteins and peptides identified by FASP method, improved FASP method and in-solution enzymatic digestion method are compared. The results show that the three different lysis buffers are suitable for the improved FASP method, and the number of proteins and peptides identified by the improved FASP method is increased by about 7%~9%, 30%~40% compared with the FASP method, which is equivalent to the in-solution enzymatic digestion method. At the same time, the improved FASP method detects more peptides with abundance greater than 10 4 6 The improved FASP method detects more peptides with abundance greater than 10 6 and better reproducibility. In addition, the improved FASP method identifies protein molecular weight and isoelectric point distribution without bias, which is universal. Finally, the optimal protein column load of the improved FASP method is determined to be 20 μg, and the method can also be used for sample pretreatment of trace protein samples (5 μg).

[0051] The results show that the adsorption of the regenerated cellulose membrane to the protein sample can be reduced and the number of identified proteins and peptide segments can be increased by grafting the amphiphilic small molecules to improve the hydrophilicity of the regenerated cellulose membrane, the sample preparation effect of the protein sample is improved, especially for the precious trace sample, the protein loss in the sample pretreatment step is reduced, and more proteins and peptide segments can be identified. The method is expected to provide help for the screening of disease-related biomarkers, the mechanism of drug efficacy, the identification of effect target points, and the diagnosis, monitoring and treatment of diseases.

[0052] The above is only used for describing the technical idea of the present application, and cannot be used to limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.

Claims

1. A method for preparing a modified ultrafiltration membrane, characterized in that: include: The regenerated cellulose membrane is immersed in an alkaline solution for activation treatment, the activated membrane material is washed, and then further immersed in a buffer solution containing amphoteric small molecules, and oscillated at 40-55°C for 8-16 hours to graft-modify the surface of the membrane material, and then dried to obtain a modified ultrafiltration membrane.

2. The method for preparing a modified ultrafiltration membrane according to claim 1, wherein The regenerated cellulose membrane was cleaned before use, including soaking the regenerated ultrafiltration membrane in isopropyl alcohol for 45-60 minutes and then rinsing it with water.

3. The method for preparing a modified ultrafiltration membrane according to claim 1, wherein The alkaline solution is a NaOH solution with a mass concentration of 4%-8%.

4. The method for preparing a modified ultrafiltration membrane according to claim 1, wherein The soaking activation treatment time is 1-2 h.

5. The method for preparing a modified ultrafiltration membrane according to claim 1, wherein The amphiphilic small molecule is 1 mol / L glycine.

6. A modified ultrafiltration membrane prepared by the method for preparing a modified ultrafiltration membrane according to any one of claims 1 to 5, characterized in that: The amount of protein adsorption on the membrane surface decreased after treatment with the modified ultrafiltration membrane.

7. An ultrafiltration centrifuge tube, characterized in that: Prepared from the modified ultrafiltration membrane described in claim 6.

8. A method for preparing protein samples for mass spectrometry analysis by low adsorption, characterized in that: The protein sample to be analyzed is lysed and incubated, and then transferred to the ultrafiltration centrifuge tube according to claim 7 for processing.

9. The method for preparing protein samples for mass spectrometry analysis by low adsorption according to claim 8, characterized in that: Used to retain proteins with a molecular weight above 10kD in the sample.

10. The method for preparing protein samples for mass spectrometry analysis by low adsorption according to claim 8, characterized in that: The optimal protein column loading capacity was 20 µg.

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