A method for detecting high-density low-abundance proteins in a body fluid

Abstract

The application discloses a detection method of high-density low-abundance proteins in body fluid, which comprises the following steps: S1) material activation, S2) material suspension, S3) sample incubation, S4) protein elution and enzymolysis, S5) pretreatment, and S6) detection. The detection method can reduce the interference of high-abundance proteins, can make the detection result more comprehensive and accurate, and fills the blank of the detection method of high-density low-abundance proteins in body fluid.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection, and particularly to a method for detecting high-density low-abundance proteins in body fluid. BACKGROUND

[0002] The proteins in human body fluid can reflect a person's physiological and pathological conditions, and detecting the body fluid proteins can diagnose various physiological and pathological conditions. In the existing body fluid protein detection methods, high-throughput proteomics quantitative analysis based on mass spectrometry is one of the preferred schemes for detection at present, but it is subject to the large fluctuation range of body fluid protein abundance. Traditional mass spectrometry detection methods can only detect hundreds of high-abundance proteins in body fluid, and cannot accurately detect and reflect the change level of the body fluid proteome, such as:

[0003] [1] Geyer, P. E., Holdt, L. M., Teupser, D. & Mann, M. Revisiting biomarker discovery by plasma proteomics. Mol. Syst. Biol. 13, 942 (2017);

[0004] [2] Geyer, P. E. et al. Plasma proteome profiling to assess human health and disease. Cell Syst. 2, 185-195 (2016);

[0005] [3] Crutchfield, C. A., Thomas, S. N., Sokoll, L. J. & Chan, D. W. Advances in mass spectrometry-based clinical biomarker discovery. Clin. Proteom. 13, 1 (2016);

[0006] [4] Keshishian, H. et al. Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry. Nat. Protoc. 12, 1683-1701 (2017);

[0007] Cao, Z., Tang, H.-Y., Wang, H., Liu, Q. & Speicher, D. W. Systematic comparison of fractionation methods for in-depth analysis of plasma proteomes. J. Proteome Res. 11, 3090-3100 (2012);

[0008] Gillette, M. A. & Carr, S. A. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat. Methods 10, 28-34 (2013).

[0009] Picotti, P., Bodenmiller, B. & Aebersold, R. Proteomics meets the scientific method. Nat. Methods 10, 24-27 (2013);

[0010] From the above listed documents, it can be seen that the traditional mass spectrometry method can only detect hundreds of high abundance proteins in body fluid (as in documents 4-7), and cannot comprehensively and accurately reflect the change level of the body fluid proteome, especially the real situation of high-density low-abundance proteins, which are often important indicators of physiological and pathological states. Therefore, the detection of this group of proteins in body fluid proteins is conducive to a deeper understanding of the occurrence and development of diseases. In view of this, there is an urgent need for a method that can detect high-density low-abundance proteins in body fluid, so as to better apply it in body fluid protein detection and acquisition of physiological and pathological related index information. SUMMARY

[0011] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a method for more comprehensive detection of high-density low-abundance proteins in body fluid.

[0012] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0013] A method for detecting high-density low-abundance proteins in body fluid, comprising the following steps:

[0014] S1) material activation: the nanoscale molecular sieve is activated by using an activator for 10-20 min, the activator comprises the following components in percentage by weight: acetonitrile 0-20%, methanol 60-80%, ethanol 0-30%, water 0-40%; the mass ratio of the activator to the nanoscale molecular sieve is 10-6000:1;

[0015] S2) material suspension: the nanoscale molecular sieve treated in the step S1 is added into a first buffer solution to form a suspension by shaking; the mass ratio of the nanoscale molecular sieve to the first buffer solution is 1:1-100;

[0016] S3) sample incubation: a body fluid sample to be detected is added into the suspension obtained in the step S2, and after being mixed uniformly, the mixture is incubated at a temperature of 37±1℃ for 5-30 min by shaking to obtain an incubation solution; the volume ratio of the body fluid sample to the suspension is Vbody fluid sample:V suspension =1:2-50;

[0017] S4) protein elution and enzymolysis: the incubation solution obtained in the step S3 is centrifuged for 5-45 min, the supernatant is discarded, and the precipitate is added into a second buffer solution and incubated at 90±5℃ for 10-30 min by shaking, the mass ratio of the precipitate to the second buffer solution is 1:2-50; then trypsin is added and enzymolysis is carried out at a temperature of 28-40℃ for 1-3 h, then trypsin is added again and enzymolysis is carried out at a temperature of 30-40℃ for 1-3 h, then formic acid or acetic acid is added to terminate the enzymolysis to obtain an enzymolysis solution;

[0018] S5) pretreatment: the enzymolysis solution obtained in the step S4 is subjected to desalination and dewatering in sequence to obtain dewatered peptides;

[0019] S6) detection: the dewatered peptides obtained in the step S5 are redissolved, and then subjected to LC-MS protein spectrum detection.

[0020] In the present application, it is further preferred that the nanoscale molecular sieve in the step S1 is one or a combination of two or more of FAU, EMT, CHA, MOR, MFI, LTL, LTA and FER nanoscale molecular sieves, and the particle size of the nanoscale molecular sieve is 5-1000 nm; further preferably, the particle size is 20-800 nm; more preferably, the particle size is 50-600 nm.

[0021] In the present application, it is further preferred that the silicon-aluminum ratio of the nanoscale molecular sieve is 1:1-2000.

[0022] In the present application, further preferred is that the first buffer solution in step S2 comprises 0-50 mmol / L urea, 0-30 mmol / L Tris-HCl, 0-200 mmol / L sodium citrate, 1-10 mmol / L EDTA, 100-300 mmol / L KCl, 100-500 mmol / L NaCl, and 0-5% CHAPS by weight percentage, and the rest is mass spectrometry grade water, and the pH value of the first buffer solution is 6-8.

[0023] In the present application, further preferred is that the silica-alumina ratio of the nanometer molecular sieve is 1:1-2000, further preferably 1:1-1000, and more preferably 1:1-500.

[0024] In the present application, further preferred is that in step S4, centrifugation is performed at a centrifugal force of 5000-10000g for 5-20 min.

[0025] In the present application, further preferred is that the second buffer solution in step S4 comprises 10-400 mmol / L DTT, 10-40 mmol / L IAA, and water and NH4HCO3, the mass of the water is m1, the weight of the NH4HCO3 is m2, m1:m2:=90-99:1-10, and the pH value of the second buffer solution is 7-9.

[0026] In the present application, further preferred is that in step S4, after the second buffer solution is added to the precipitate and the precipitate is shaken and incubated, the protein content in the precipitate is detected before trypsin is added for enzymolysis.

[0027] In the present application, further preferred is that in step S4, the weight ratio of the trypsin added for enzymolysis to the protein in the precipitate is 1:30-100.

[0028] In the present application, further preferred is that in step S5, the enzymolysis solution is subjected to desalination treatment by using a C18 column.

[0029] Compared with the prior art, the beneficial effects of the present application are that by using activated nanoscale molecular sieves to enrich "low-abundance high-density proteins" in body fluids, and then eluting residual high-abundance proteins by a buffer system of different salt ions, a first buffer with a pH value of 6-8, and a second buffer with a pH value of 7-9, reducing the interference of high-abundance proteins on detection, and then using liquid chromatography mass spectrometry to detect body fluid proteins, compared with the existing method of directly detecting proteins in body fluids by mass spectrometry, the number and types of proteins detected are greatly improved. In addition, this method selectively enriches low-abundance high-density proteins, fills the gap in the field of body fluid protein detection, and can be widely applied to scientific research and medical fields. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The electrophoresis graph (protein gel graph) of the plasma sample in P1 group in Example 1 and the electrophoresis detection after the treatment in Example 1;

[0031] Figure 2 The XRD detection graph of the nanoscale molecular sieve in Example 1;

[0032] Figure 3 The SEM scanning electron microscope scanning graph of the nanoscale molecular sieve in Example 1;

[0033] Figure 4 The TEM scanning electron microscope scanning graph of the nanoscale molecular sieve in Example 1. IMPLEMENTATION

[0034] The present application will be further described below in conjunction with the specific embodiments and the accompanying drawings of the specification. It should be noted that the following described embodiments or technical features can be combined in any manner to form new embodiments without conflict. Except for special explanations, the materials and equipment used in the embodiments can be purchased from the market. The specific embodiments are exemplary and are used to explain the present application, but cannot be understood as limiting the scope of protection of the present application.

[0035] A detection method of high-density low-abundance proteins in body fluids, comprising the following steps:

[0036] S1) Material activation: using an activator to activate the nanoscale molecular sieve for 10-20 min, the activator comprising the following components by weight percentage: acetonitrile 0-20%, methanol 60-80%, ethanol 0-30%, water 0-40%; the mass ratio of the activator to the nanoscale molecular sieve is 10-6000:1;

[0037] S2) Material suspension: adding the nanoscale molecular sieve treated in S1 step into the first buffer to form a suspension by shaking; the mass ratio of the nanoscale molecular sieve to the first buffer is 1:1-100;

[0038] S3) detecting sample incubation: adding the body fluid sample to be detected into the suspension liquid obtained in step S2, mixing uniformly, and then incubating at a temperature of 37±1 DEG C for 5-30 min to obtain an incubation liquid; the volume ratio of the body fluid sample to the suspension liquid is V body fluid sample: V suspension liquid = 1:2-50;

[0039] S4) protein elution and enzymolysis: centrifuging the incubation liquid obtained in step S3 for 5-45 min, discarding the supernatant, and taking the precipitate, adding the precipitate into a second buffer liquid, and incubating at 90±5 DEG C for 10-30 min, the mass ratio of the precipitate to the second buffer liquid is 1:2-50; then adding trypsin, and enzymolyzing at a temperature of 28-40 DEG C for 1-3 h, then adding trypsin again, and enzymolyzing at a temperature of 30-40 DEG C for 1-3 h, and then adding formic acid or acetic acid to terminate the enzymolysis, to obtain an enzymolysis liquid;

[0040] S5) pre-treatment: sequentially performing desalination and dewatering treatment on the enzymolysis liquid obtained in step S4 to obtain dewatered peptides;

[0041] S6) detection: redissolving the dewatered peptides obtained in step S5, and then performing LC-MS protein spectrum detection.

[0042] By using the activated nanoscale molecular sieve to enrich the "low-abundance high-density protein" in the body fluid, then eluting the residual high-abundance protein by using a buffer system of the first buffer liquid with different salt ions and a pH value of 6-8 and the second buffer liquid with a pH value of 7-9, reducing the interference of the high-abundance protein on the detection, and then detecting the body fluid protein by using the liquid chromatography mass spectrometry, compared with the existing method of directly detecting the protein in the body fluid by using the mass spectrometry, the number and the type of the detected proteins are greatly improved, in addition, the low-abundance high-density protein is selectively enriched, the blank in the field of body fluid protein detection is filled, and the method can be widely applied to the fields of scientific research and medical treatment.

[0043] The body fluid in the application refers to the total of water in the body and various substances dispersed in the water, including but not limited to whole blood, plasma, serum, urine, cerebrospinal fluid, intestinal lavage fluid, etc.

[0044] In the application, it is further preferred that the nanoscale molecular sieve in step S1 is one or a combination of more than two of FAU, EMT, CHA, MOR, MFI, LTL, LTA and FER nanoscale molecular sieves; the particle size of the nanoscale molecular sieve is 5-1000 nm; the further preferred particle size is 20-800 nm; and the more further preferred particle size is 50-600 nm.

[0045] In this invention, a further preferred embodiment is that the silicon-to-aluminum ratio of the nano-molecular sieve is 1:1-2000; the nano-molecular sieve can be selected as a nano-molecular sieve with the following structural formula: (Na+)O·Al2O3·ySiO2·yH2O.

[0046] Due to limitations in sensitivity and resolution of current mass spectrometry technology and equipment, previous body fluid tests have been affected by high-abundance proteins, preventing existing methods from comprehensively and accurately detecting the proteome of body fluids. To further reduce the impact of high-abundance proteins (such as hemoglobin) on the test results and improve the comprehensiveness and accuracy of the detection, a preferred embodiment is that the first buffer solution in step S2 comprises: 0-50 mmol / L urea, 0-30 mmol / L Tris-HCl (TRIS hydrochloride (NH2C(CH2OH)3 · HCl)), 0-200 mmol / L sodium citrate, 1-10 mmol / L EDTA, 100-300 mmol / L KCl, 100-500 mmol / L NaCl, and 0-5% by weight CHAPS (3-[3-(cholanamidopropyl)dimethylamino]propanesulfonic acid hydrate (C 32 H 58 The first buffer solution consists of N2O7S·xH2O, with the remainder being mass spectrometry grade water, and has a pH of 6-8. The second buffer solution in step S4 comprises 10-400 mmol / L DTT, 10-40 mmol / L IAA, water, and NH4HCO3 at a molar volume ratio of m1 and m2, with m1:m2 = 90-99:1-10, and has a pH of 7-9.

[0047] In this invention, a further preferred embodiment is that the silicon-to-aluminum ratio of the nanomolecular sieve is 1:1-2000; a further preferred embodiment is that the silicon-to-aluminum ratio is 1:1-1000; and an even more preferred embodiment is that the silicon-to-aluminum ratio is 1:1-500.

[0048] In this invention, the magnitude and time of centrifugal force can be selected as described in step S4, where centrifugation is performed for 5-20 minutes at a centrifugal force of 5000-10000g.

[0049] In a further preferred embodiment of the present invention, the second buffer solution in step S4 comprises 10-400 mmol / L DTT (DL-dithiothreitol (HSCH2CH(OH)CH(OH)CH2SH)), 10-40 mmol / L IAA (iodoacetamide (ICH2CONH2)), water, and NH4HCO3, wherein the mass of the water is m1, the weight of the NH4HCO3 is m2, m1:m2:=90-99::1-10, and the pH value of the second buffer solution is 7-9.

[0050] To improve enzymatic hydrolysis efficiency and avoid wasting protease, the protein content in the precipitate can be detected before adding the second buffer solution. After adding the second buffer solution and shaking and incubating the precipitate, the protein content in the precipitate can be detected before adding trypsin for enzymatic hydrolysis. Then, based on the measured protein content in the precipitate, in step S4, the weight ratio of trypsin added for enzymatic hydrolysis to protein in the precipitate is 1:30-100.

[0051] For the desalting process in this invention, a C18 column is used to desalt the enzymatic hydrolysate in step S5; for the drying process, vacuum drying can be used. Example

[0052] A method for detecting high-density, low-abundance proteins in body fluids, wherein the body fluid is plasma, includes the following steps:

[0053] S1) Material activation: The nanoscale molecular sieve is activated for 10 minutes using an activating agent, which comprises the following components by weight percentage: acetonitrile 20%, methanol 60%, ethanol 10%, and water 10%; the mass ratio of the activating agent to the nanoscale molecular sieve is 100:1; the nanoscale molecular sieve used in step S1 is an FAU molecular sieve with a silicon-to-aluminum ratio of 2.

[0054] S2) Material suspension: The nanoscale molecular sieve treated in step S1 is added to the first buffer solution and shaken to form a suspension; the mass ratio of the nanoscale molecular sieve to the first buffer solution is 1:100.

[0055] The first buffer solution in step S2 comprises: 10 mmol / L urea, 15 mmol / L Tris-HCl (TRIS hydrochloride (NH2C(CH2OH)3 · HCl)), 10 mmol / L sodium citrate, 5 mmol / L EDTA, 200 mmol / L KCl, 400 mmol / L NaCl, and 4% by weight CHAPS (3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid hydrate (C 32 H58 The buffer solution consists of N2O7S·xH2O, with the remainder being mass spectrometry grade water, and the pH value of the first buffer solution is 7.8.

[0056] S3) Sample incubation: Add the body fluid sample to be tested to the suspension obtained in step S2, mix well, and incubate with shaking at 37°C for 30 minutes to obtain the incubation solution; the volume ratio of the body fluid sample to the suspension is: V body fluid sample : V suspension = 1:20;

[0057] S4) Protein elution and enzymatic digestion: Centrifuge the incubation solution obtained in step S3 for 10 min, discard the supernatant, take the precipitate, add the precipitate to the second buffer and incubate at 90°C with shaking for 30 min. The mass ratio of the precipitate to the second buffer is 1:20. Then add trypsin (TE Trypsin from Gibgo) and digest at 37°C for 3 h. Then add trypsin again and digest at 37°C for 3 h. Finally, add formic acid or acetic acid to terminate the digestion and obtain the enzymatic digest.

[0058] In step S4), after adding a second buffer to the precipitate and incubating it with shaking at 95°C, the protein content is detected before adding trypsin for enzymatic digestion. The protein content is quantified using the BCA protein quantification method (Thermo Scientific™ Pierce™ BCA Protein Detection Kit - Reducing Agent Compatible). The ratio of the amount of trypsin added to the protein content in the precipitate is 1:50.

[0059] The second buffer solution in step S4 comprises 100 mmol / L DTT, 20 mmol / L IAA (by molar volume), water, and NH4HCO3, wherein the mass of the water is m1, the weight of the NH4HCO3 is m2, m1:m2:=90:10, and the pH of the second buffer solution is 9.0.

[0060] S5) Pretreatment: The enzymatic hydrolysate obtained in step S4 was subjected to desalting and drying treatments. Desalting: The solution obtained in step S5 was added to a C18 column (Pierce™ Peptide Desalting Spin Columns), and the column was aspirated at 10 kPa for 10 min using a negative pressure pump. The filtered solution was then collected. Drying: The collected filtered solution was placed in a vacuum concentrator (Eppendorf Concentrator plus) and aspirated at room temperature for 4 h to obtain the dried peptides.

[0061] S6) Detection: The dried peptide obtained in step S5 was reconstituted and then subjected to LC-MS protein spectrometry (ThermoFisher Orbitrap Exploris 480 mass spectrometer).

[0062] Five plasma samples were collected (referred to as P1, P2, P3, P4, and P5, respectively), from five recruited volunteers. The samples were tested using the methods described above, and repeatability experiments were performed. The quantities of peptides and proteins in the five plasma samples are shown in the table below.

[0063]

[0064] As can be seen from the data in the table above, the number of peptides identified in each sample exceeded 20,000 and the number of protein types exceeded 3,500. Furthermore, the data obtained from the repeatability experiments were similar, reflecting the stability of the detection method of this invention. It is evident that the detection method of this invention can comprehensively and accurately reflect the quantity and index level of high-density, low-abundance proteins in body fluids (plasma).

[0065] Plasma proteins from group P1 were directly detected by electrophoresis (protein gel detection) and compared with the sample processed by the method in Example 1 of this invention. See [link to example]. Figure 1 As can be seen from the comparison of the boxes in the figure, after the treatment of the present invention, the high abundance protein in the plasma is significantly reduced and the high density protein content is significantly increased. It can be seen that the detection method of the present invention can effectively reduce the interference of high abundance proteins and improve the comprehensiveness of low abundance protein detection.

[0066] For the nanomolecular sieve in this embodiment, its XRD pattern and scanning electron microscope image are shown below. Figures 2-4 .

[0067] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting high-density, low-abundance proteins in body fluids, characterized in that... Includes the following steps: S1) Material activation: The nanoscale molecular sieve is activated for 10-20 minutes using an activating agent, which comprises the following components by weight percentage: acetonitrile 0-20%, methanol 60-80%, ethanol 0-30%, and water 0-40%; the mass ratio of the activating agent to the nanoscale molecular sieve is 10-6000:

1. S2) Material suspension: The nanoscale molecular sieve treated in step S1 is added to the first buffer solution and shaken to form a suspension; the mass ratio of the nanoscale molecular sieve to the first buffer solution is 1:1-100. S3) Sample incubation: Add the body fluid sample to be tested to the suspension obtained in step S2, mix well, and incubate with shaking at 37±1℃ for 5-30 minutes to obtain the incubation solution; the volume ratio of the body fluid sample to the suspension is: V body fluid sample : V suspension = 1 : 2-50; S4) Protein elution and enzymatic digestion: Centrifuge the incubation solution obtained in step S3 for 5-45 min, discard the supernatant, take the precipitate, add the precipitate to the second buffer and incubate with shaking at 90±5℃ for 10-30 min. The mass ratio of the precipitate to the second buffer is 1:2-50. Then add trypsin and digest at 28-40℃ for 1-3 h. Then add trypsin again and digest at 30-40℃ for 1-3 h. Finally, add formic acid or acetic acid to terminate the digestion and obtain the enzymatic digest. S5) Pretreatment: The enzymatic hydrolysate obtained in step S4 is subjected to desalting and drying treatment in sequence to obtain dried peptide fragments. S6) Detection: The dried peptide obtained in step S5 is reconstituted and then subjected to LC-MS protein spectroscopy detection. The first buffer solution in step S2 comprises: 0-50 mmol / L urea, 0-30 mmol / L Tris-HCl, 0-200 mmol / L sodium citrate, 1-10 mmol / L EDTA, 100-300 mmol / L KCl, 100-500 mmol / L NaCl, and 0-5% CHAPS by weight, with the balance being mass spectrometry grade water. The pH value of the first buffer solution is 6-8. The second buffer solution in step S4 comprises 10-400 mmol / L DTT, 10-40 mmol / L IAA, water, and NH4HCO3 at a molar volume ratio of m1 and m2, with a ratio of m1:m2:=90-99:1-10. The pH of the second buffer solution is 7-9.

2. The detection method according to claim 1, characterized in that, The nanoscale molecular sieve in step S1 is one or a combination of two or more of FAU, EMT, CHA, MOR, MFI, LTL, LTA and FER nanoscale molecular sieves, and the particle size of the nanoscale molecular sieve is 5 to 1000 nm.

3. The detection method according to claim 2, characterized in that, The silicon-to-aluminum ratio of the nanomolecular sieve is 1:1-2000.

4. The detection method according to claim 1, characterized in that, In step S4, centrifugation is performed for 5-20 minutes at a centrifugal force of 5000-10000g.

5. The detection method according to claim 1, characterized in that, In step S4, after adding the second buffer solution to the precipitate and shaking it for incubation, and before adding trypsin for enzymatic digestion, the protein content in the precipitate is detected.

6. The detection method according to claim 5, characterized in that, In step S4, the weight ratio of trypsin added for enzymatic hydrolysis to protein in the precipitate is 1:30-100.

7. The detection method according to claim 1, characterized in that, In step S5, a C18 column is used to desalt the enzymatic hydrolysate.

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