A method for detecting a whole blood proteome

Abstract

The application discloses a kind of detection methods of whole blood proteome, comprising the following steps: S1) material activation, S2) material suspension, S3) detection sample incubation, S4) protein elution and enzymolysis, S5) pretreatment, S6) detection.The detection method of the application can make the result of detection more comprehensive and accurate, and the number of protein species detected by single sample is up to 4000 or more, filling the blank of whole blood proteome detection method.

Description

Technical Field

[0001] This invention relates to the field of detection, specifically to a method for detecting whole blood protein profiles. Background Technology

[0002] Human blood proteins can reflect a person's physiological and pathological conditions, and detecting blood proteins can help diagnose various physiological and pathological conditions. Among existing blood sample testing methods, high-throughput proteomics quantitative analysis based on mass spectrometry is currently one of the preferred solutions. However, due to limitations in the sensitivity and resolution of this technology, the detection of blood proteins can be interfered with by high-abundance proteins such as hemoglobin, causing other relatively low-abundance proteins to remain undetected, such as:

[0003] [1] Chambers AG, Percy AJ, Hardie DB, Borchers CH. Comparison of proteins in whole blood and dried blood spot samples by LC / MS / MS. J Am SocMass Spectrom. 2013 Sep;24(9):1338-45. doi: 10.1007 / s13361-013-0678-x. Epub2013 Jul 3. PMID: 23821375;

[0004] [2] Molloy MP, Hill C, O'Rourke MB, Chandra J, Steffen P, McKay MJ,Pascovici D, Herbert BR. Proteomic Analysis of Whole Blood Using VolumetricAbsorptive Microsampling for Precision Medicine Biomarker Studies. J ProteomeRes. 2022 Apr 1;21(4):1196-1203. doi: 10.1021 / acs.jproteome.1c00971. Epub2022 Feb 15. PMID: 35166117;

[0005] [3] Mc Ardle A, Binek A, Moradian A, Chazarin Orgel B, Rivas A, Washington KE, Phebus C, Manalo DM, Go J, Venkatraman V, Coutelin Johnson CW, Fu Q, Cheng S, Raedschelders K, Fert-Bober J, Pennington SR, Murray CI, VanEyk JE. Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids. Clin Chem. 2022 Mar 4;68(3):450-460. doi: 10.1093 / clinchem / hvab202. PMID: 34687543;

[0006] [4] Kashirina DN, Brzhozovskiy AG, Sun W, Pastushkova LK, Popova OV,Rusanov VB, Nikolaev EN, Larina IM, Kononikhin AS. Proteomic Characterization of Dry Blood Spots of Healthy Women During Simulation the MicrogravityEffects Using Dry Immersion. Front Physiol. 2022 Jan 11;12:753291. doi:10.3389 / fphys.2021.753291. PMID: 35087415; PMCID: PMC8787266;

[0007] As can be seen from the literature listed above, existing quantitative analysis of blood proteomics is limited to plasma detection, and existing whole blood protein detection methods can only detect 300-1400 proteins. Moreover, the sample preparation and instrument loading time often exceeds 48 hours, which affects the accuracy of the detection results.

[0008] While plasma testing can be applied to high-throughput proteomics quantitative analysis based on mass spectrometry to some extent, reflecting the physiological and pathological conditions of the tested subjects, a significant amount of blood proteins are lost during the preparation of the plasma sample. This means the test results cannot fully reflect the protein content in the blood, and therefore cannot objectively and completely reflect the physiological and pathological conditions. In view of this, there is an urgent need for a method to detect whole blood proteins, so as to be better applied in blood protein detection and the acquisition of physiological and pathological related indicators. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for more comprehensive detection of whole blood proteome.

[0010] To achieve the above objectives, the present invention employs the following technical solution:

[0011] A method for detecting whole blood proteome includes the following steps:

[0012] 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: 60-80% methanol, 0-30% ethanol, and 0-40% water; the mass ratio of the activating agent to the nanoscale molecular sieve is 100-5000:1.

[0013] 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 1:2-50.

[0014] S3) Sample incubation: Add the whole blood 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 whole blood sample to the suspension is: V_whole_blood_sample:V_suspension = 0.1-2:1;

[0015] S4) Protein elution and enzymatic digestion: Centrifuge the incubation solution obtained in step S3 for 5-30 min, discard the supernatant, take the precipitate, add the precipitate to the second buffer and incubate with shaking at 95±2℃ for 10-30 min. The mass ratio of the precipitate to the second buffer is 1:2-50. Then add trypsin and digest at 30-40℃ for 1.8-2.2 h. Then add trypsin again and digest at 30-40℃ for 1.8-2.2 h. Finally, add formic acid or acetic acid to terminate the digestion and obtain the enzymatic digest.

[0016] S5) Pretreatment: The enzymatic hydrolysate obtained in step S4 is subjected to desalting and drying treatment in sequence to obtain dried peptide fragments.

[0017] S6) Detection: The dried peptide obtained in step S5 is reconstituted and then subjected to LC-MS protein spectroscopy detection.

[0018] In this invention, a further preferred embodiment is 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 5~1000 nm; a further preferred particle size is 20-800 nm, and an even more preferred particle size is 50-600 nm.

[0019] 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.

[0020] In a further preferred embodiment of the present invention, the first buffer solution in step S2 comprises: 0-30 mmol / L Tris-HCl, 1-10 mmol / L EDTA, 100-300 mmol / L KCl, 100-500 mmol / L NaCl, and 0-5% CHAPS by weight, with the remainder being mass spectrometry grade water, and the pH value of the first buffer solution is 6-8.

[0021] In this invention, a further preferred embodiment is that the whole blood sample to be tested in step S3 is stabilized before the suspension is added: a stabilizing solution is added to the sample to be tested, and then it is frozen at a temperature of -20°C to -80°C for more than 60 minutes, and then dissolved at room temperature; the stabilizing solution contains the following solutes in the following mass-volume ratios: 0-0.05 g / 100 ml of NaCl, 0-0.3 g / 100 ml of KCl, and the stabilizing solution contains the following in the following weight percentages: 0-40% H2O, 60%-100% ethanol.

[0022] In this invention, a further preferred embodiment is that in step S4, centrifugation is performed for 10 minutes at a centrifugal force of 10000g.

[0023] In a further preferred embodiment of the present invention, 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, 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.

[0024] In a further preferred embodiment of the present invention, in step S4, after adding the second buffer solution to the precipitate and shaking and incubating, and before adding trypsin for enzymatic hydrolysis, the protein content in the precipitate is detected.

[0025] In this invention, a further preferred embodiment is that, in step S4, the weight ratio of the trypsin added for enzymatic hydrolysis to the protein in the precipitate is 1:30-100.

[0026] In this invention, a further preferred embodiment is that a C18 column is used to desalt the enzymatic hydrolysate in step S5.

[0027] Compared to existing technologies, the advantages of this invention lie in its ability to indiscriminately enrich all proteins in whole blood using activated nanoscale molecular sieves, forming a soft protein crown on the material surface. This enrichment process is more sensitive to low-abundance proteins in whole blood, avoiding interference caused by excessive enrichment of high-abundance proteins (such as hemoglobin) in previous methods. Furthermore, the accompanying gradient buffer system further elutes residual high-abundance proteins, resulting in more comprehensive and accurate detection results and a significant improvement in detection depth. Using this method, over 4000 protein types can be detected in a single sample within 12 hours. In contrast, existing whole blood protein detection methods can only detect 300-1400 proteins, and sample preparation and processing time often exceeds 48 hours. This method fills a gap in whole blood proteomics detection methods and can be widely applied in scientific research and medical fields. Attached Figure Description

[0028] Figure 1 This is a graph showing the peptide identification data from the six whole blood samples tested in Example 1;

[0029] Figure 2 This is a graph showing the protein identification data from the six whole blood samples tested in Example 1;

[0030] Figure 3 This is the first-order mass spectrum of peptides from the whole blood sample in group P1 of Example 1;

[0031] Figure 4 This is a secondary mass spectrometry image of peptide fragments from the whole blood sample in group P1 of Example 1;

[0032] Figure 5 This is a graph showing the correlation data of repeatability experiments for group P1 in Example 1. Implementation

[0033] The present invention will now be further described in conjunction with specific embodiments and accompanying drawings. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the materials and equipment used in this embodiment are commercially available. The specific embodiments are exemplary and are only used to explain this application, and should not be construed as limiting the scope of protection of this application.

[0034] A method for detecting whole blood proteome includes the following steps:

[0035] 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: 60-80% methanol, 0-30% ethanol, and 0-40% water; the mass ratio of the activating agent to the nanoscale molecular sieve is 100-5000:1.

[0036] 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 1:2-50.

[0037] S3) Sample incubation: Add the whole blood 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 whole blood sample to the suspension is: V_whole_blood_sample:V_suspension = 0.1-2:1;

[0038] S4) Protein elution and enzymatic digestion: Centrifuge the incubation solution obtained in step S3 for 5-30 min, discard the supernatant, take the precipitate, add the precipitate to the second buffer and incubate with shaking at 95±2℃ for 10-30 min. The mass ratio of the precipitate to the second buffer is 1:2-50. Then add trypsin and digest at 30-40℃ for 1.8-2.2 h. Then add trypsin again and digest at 30-40℃ for 1.8-2.2 h. Finally, add formic acid or acetic acid to terminate the digestion and obtain the enzymatic digest.

[0039] S5) Pretreatment: The enzymatic hydrolysate obtained in step S4 is subjected to desalting and drying treatment in sequence to obtain dried peptide fragments.

[0040] S6) Detection: The dried peptide obtained in step S5 is reconstituted and then subjected to LC-MS protein spectroscopy detection.

[0041] By employing activated nanoscale molecular sieves to indiscriminately enrich all proteins in whole blood, the protein loss caused during plasma preparation in previous detection methods is avoided. Then, a matching buffer system of first and second buffer components reduces high-abundance proteins (such as hemoglobin), thereby making the detection results more comprehensive and accurate. Through the method of this invention, the number of protein types detected in a single sample can reach more than 4,000, filling the gap in whole blood proteomics detection methods and can be widely applied in scientific research and medical fields.

[0042] In this invention, a further preferred embodiment is 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 5~1000 nm; a further preferred particle size is 20-800 nm, and an even more preferred particle size is 50-600 nm.

[0043] In this invention, a further preferred embodiment is that the silicon-to-aluminum ratio of the nano-molecular sieve is 1:1-2000; a further preferred embodiment is 1:1-1000; and an even more preferred embodiment is 1:1-500. The nano-molecular sieve can be selected from nano-molecular sieves with the following structural formula: (Na+)O·Al2O3·ySiO2·yH2O.

[0044] Due to limitations in sensitivity and resolution of current mass spectrometry technology and equipment, previous whole blood tests have been affected by high-abundance proteins, preventing existing methods from comprehensively and accurately detecting the whole blood proteome. To further reduce the impact of high-abundance proteins (such as hemoglobin) on test results and improve the comprehensiveness and accuracy of the detection, the first buffer in step S2 includes: 0-30 mmol / L Tris-HCl (TRIS hydrochloride (NH2C(CH2OH)3 · HCl)), 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 58The first buffer solution consists of N2O7S·xH2O), with the remainder being mass spectrometry grade water, and the pH value of the first buffer solution is 6-8. The second buffer solution in step S4 includes 10-400 mmol / L of DTT (DL-dithiothreitol (HSCH2CH(OH)CH(OH)CH2SH)), 10-40 mmol / L of IAA (iodoacetamide (ICH2CONH2)), water, and NH4HCO3, with the mass of the water being m1 and the weight of the NH4HCO3 being m2, m1:m2:=90-99::1-10, and the pH value of the second buffer solution is 7-9.

[0045] To avoid the instability of blood's physicochemical properties affecting the test results and to improve the accuracy and stability of the test results, the whole blood sample to be tested in step S3 is stabilized before adding the suspension: a stabilizing solution is added to the sample to be tested, and then it is frozen at a temperature of -20℃ to -80℃ for more than 60 minutes, and then dissolved at room temperature; the stabilizing solution contains the following solutes by mass-volume ratio: 0-0.05g / 100ml of NaCl, 0-0.3g / 100ml of KCl, and the stabilizing solution contains by weight percentage: 0-40% H2O, 60%-100% ethanol.

[0046] In this invention, regarding the magnitude of the centrifugal force, step S4 involves centrifuging for 10 minutes at a centrifugal force of 10000g.

[0047] 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.

[0048] 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

[0049] A method for detecting whole blood proteome includes the following steps:

[0050] S1) Material activation: The nanoscale molecular sieve is activated for 20 minutes using an activating agent, which comprises the following components by weight percentage: 60% methanol, 10% ethanol, and 30% water; the nanoscale molecular sieve used in step S1 is an FAU molecular sieve with a silicon-to-aluminum ratio of 2 and an average particle size of 80 nm; the mass ratio of the activating agent to the nanoscale molecular sieve is 200:1.

[0051] S2) Material suspension: The nanoscale molecular sieves treated in step S1 are added to the first buffer solution and shaken to form a suspension; the first buffer solution in step S2 includes: 20 mmol / L Tris-HCl, 10 mmol / L EDTA, 100 mmol / L KCl, 200 mmol / L NaCl, and 5% CHAPS by weight, with the remainder being mass spectrometry grade water, and the pH value of the first buffer solution is 7.5;

[0052] S3) Sample incubation: Add the whole blood sample to be tested to the suspension obtained in step S2, mix well, and incubate with shaking at 37°C for 20 minutes to obtain the incubation solution; the volume ratio of the whole blood sample to the suspension is: V_whole_blood_sample : V_suspension = 1:10; the whole blood sample to be tested in step S3 is stabilized before being added to the suspension: add stabilizing solution to the sample to be tested, and then... At -80℃ The solution is frozen at a temperature of 60°C or higher for at least 60 minutes, and then thawed at room temperature. The stabilized solution contains the following solutes in the following mass-volume ratios: 0.02 g / 100 ml NaCl and 0.1 g / 100 ml KCl. The stabilized solution contains the following by weight percentages: 20% H2O and 80% ethanol.

[0053] S4) Protein elution and enzymatic digestion: Centrifuge the incubation solution obtained in step S3 for 10 min, discard the supernatant, and collect the precipitate; in step S4, centrifugation is performed at 10000g for 10 min; add the precipitate obtained in step S4 to the second buffer and incubate with shaking at 95°C for 30 min; then add trypsin (TE Trypsin from Gibgo) and digest at 37°C for 2 h, then add trypsin again and digest at 37°C for 2 h, then add formic acid to terminate the digestion, obtaining the enzymatic digest; the second buffer in step S4 includes 100 mmol / L DTT by molar volume. 、 10 mmol / L IAA, water and NH4HCO3, wherein the mass of water is m1, the weight of NH4HCO3 is m2, m1:m2:=95:5, and the pH of the second buffer solution is 7.8;

[0054] 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.

[0055] 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.

[0056] 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).

[0057] Six whole blood samples were collected (referred to as P1, P2, P3, P4, P5, and P6, respectively; these six groups of blood were obtained from six recruited healthy volunteers, and blood was collected from the left elbow vein. The blood was collected using EDTA tubes, mixed thoroughly, and stored). The samples were tested according to the method described above. The protein content in the precipitate detected in step S4 was 0.237–0.386 mg / ml. Then, trypsin was added according to the ratio of trypsin to protein determined in step S4 for enzymatic digestion. The number of peptides identified under DIA for 55 minutes in each group were as follows: P1 group 46189, P2 group 45202, P3 group 45016, P4 group 44230, P5 group 45593, and P6 group 45549 (see [reference missing]). Figure 1 The number of protein types detected in each group were as follows: P1 group 4267, P2 group 4345, P3 group 4234, P4 group 4339, P5 group 4337, and P6 group 4235 (see [link to relevant documentation]). Figure 2 As can be seen, the number of protein samples detected in each group was greater than 4,000, which can comprehensively and accurately complete the whole blood protein assay.

[0058] Among them, the primary and secondary mass spectra of peptide detection in group P1 (see...) Figure 3 , Figure 4As can be seen, the method of the present invention can detect the proteome in a relatively comprehensive and accurate manner. Regarding the stability of the method, through repeatability experiments (reproducing and detecting whole blood samples from P1 patients six times), the correlation remained between 0.990 and 0.998, indicating that the detection method of the present invention has good stability.

[0059] Existing whole blood proteomics detection methods, both domestically and internationally, mainly involve direct whole blood detection or detecting whole blood after drying into blood spots. Regardless of the method, only 300-1400 proteins can be detected, far lower than the detection depth of this technology. The detection time is typically 48 hours or more, significantly longer than the required time for this technology. Therefore, this method represents a breakthrough in both detection time and detection depth compared to traditional whole blood protein detection methods, making whole blood protein detection applicable to clinical and research settings and filling a gap in this field.

[0060] 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 whole blood proteomics, 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: 60-80% methanol, 0-30% ethanol, and 0-40% water; the mass ratio of the activating agent to the nanoscale molecular sieve is 100-5000:

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 1:2-50. S3) Sample incubation: Add the whole blood 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 whole blood sample to the suspension is: V_whole_blood_sample:V_suspension = 0.1-2:1; S4) Protein elution and enzymatic digestion: Centrifuge the incubation solution obtained in step S3 for 5-30 min, discard the supernatant, take the precipitate, add the precipitate to the second buffer and incubate with shaking at 95±2℃ for 10-30 min. The mass ratio of the precipitate to the second buffer is 1:2-50. Then add trypsin and digest at 30-40℃ for 1.8-2.2 h. Then add trypsin again and digest at 30-40℃ for 1.8-2.2 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: 0-30 mmol / L Tris-HCl, 1-10 mmol / L EDTA, 100-300 mmol / L KCl, 100-500 mmol / L NaCl, and 0-5% CHAPS by weight, with the remainder 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, 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.

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 S3, the whole blood sample to be tested is stabilized before adding the suspension: a stabilizing solution is added to the sample to be tested, and then it is frozen at a temperature of -20°C to -80°C for more than 60 minutes, and then dissolved at room temperature; the stabilizing solution contains the following solutes by mass-volume ratio: 0-0.05 g / 100 ml NaCl, 0-0.3 g / 100 ml KCl, and the stabilizing solution contains by weight percentage: 0-40% H2O, 60%-100% ethanol.

5. The detection method according to claim 1, characterized in that, In step S4, centrifugation is performed for 10 minutes at a centrifugal force of 10000g.

6. 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.

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

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

Images