A method for pretreatment of plasma proteomics samples

Abstract

This invention discloses a method for pretreatment of plasma proteomics samples, comprising the following steps: Step S1, adding a small molecule drug to the plasma sample to bind the small molecule drug to albumin in the plasma, inhibiting the enzymatic digestion of albumin; Step S2, transferring the plasma sample treated in Step S1 to an ultrafiltration tube, centrifuging to remove small molecule metabolites and unbound small molecule drugs from the plasma; Step S3, adding buffer and protease to the ultrafiltration tube in Step S2 to perform enzymatic digestion of proteins. This method for pretreatment of plasma proteomics samples utilizes the interaction between small molecule drugs and albumin to form a stable folded state, thereby affecting the structure and biophysical properties of proteins, reducing the sensitivity of albumin to proteolytic enzymes, minimizing the influence of albumin on the identification of medium and low abundance proteins in plasma, and greatly improving the convenience of clinical sample analysis.

Description

Technical Field

[0001] This invention relates to the field of plasma proteomics technology, and in particular to a method for pretreatment of plasma proteomics samples. Background Technology

[0002] Most drugs bind to plasma proteins in the body to form drug-conjugated forms. Plasma proteins act as carriers responsible for the transport of endogenous and exogenous molecules in the blood. Among them, albumin and α-1-acid glycoprotein are the main drugs that bind to small molecules. Human serum albumin, as the most abundant and important plasma protein, has been extensively studied.

[0003] Recent studies on drug discovery based on chemical proteomics have shown that small molecule drugs bind to proteins to form stable folded states, thereby affecting the structure and biophysical properties of proteins, such as their thermal stability and sensitivity to proteolytic enzymes.

[0004] Liquid chromatography-mass spectrometry (LC-MS) has brought opportunities for proteomics analysis. The high efficiency of chromatographs and the rapid scanning capability of mass spectrometers have enabled the identification of more and more proteins in plasma, with the number of detected proteins increasing dramatically from hundreds to more than 10,000.

[0005] However, the complex composition of plasma and the wide range of protein concentrations present numerous challenges to plasma proteomics analysis. Human serum albumin (HSA) is the most abundant protein in plasma, accounting for 55% of the total plasma protein content, and its presence severely affects the identification of low- and medium-abundance proteins. Current solutions mainly involve using specific antibodies or immunoassay kits to remove albumin and other high-abundance proteins, or pre-fractionating peptides before subsequent processing. However, these strategies are time-consuming, labor-intensive, and expensive, making them unsuitable for analyzing large volumes of clinical samples. Therefore, we propose a method for sample pretreatment in plasma proteomics. Summary of the Invention

[0006] The main objective of this invention is to provide a method for pretreatment of plasma proteomics samples. This method utilizes the interaction between small molecule drugs and albumin to form a stable folded state, thereby affecting the structure and biophysical properties of proteins, reducing the sensitivity of albumin to proteolytic enzymes, minimizing the impact of albumin on the identification of medium and low abundance proteins in plasma, greatly improving the convenience of clinical sample analysis, and significantly reducing costs.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preprocessing plasma proteomics samples includes the following steps: Step S1: Add the small molecule drug to the plasma sample and vortex it for a period of time to simulate human body temperature and pH value, so that the small molecule drug binds to albumin in the plasma and inhibits the enzymatic degradation of albumin. Step S2: Transfer the plasma sample processed in step S1 to an ultrafiltration tube and centrifuge to remove small molecule metabolites and unbound small molecule drugs from the plasma. Step S3: Add buffer and protease to the ultrafiltration tube from step S2 to perform enzymatic digestion of the protein; Step S4: Centrifuge to collect the external peptides of the protein generated in step S4, and perform mass spectrometry analysis.

[0008] Preferably, in step S1, the plasma sample is a clinically collected human plasma sample.

[0009] Preferably, in step S1, the small molecule drug is a drug that can bind to albumin.

[0010] Preferably, in step S1, the concentration of the small molecule drug is 100mM-500mM.

[0011] Preferably, in step S1, the human body temperature is 35-38℃, the pH value is 7.0-8.0, and the vortex time is 10-40 min.

[0012] Preferably, in step S2, the ultrafiltration tube has a capacity of 0.5-5 mL and a specification of 10 kD or 30 kD.

[0013] Preferably, the enzyme digestion lasts for 30 min to 2 h.

[0014] Preferably, the buffer solution comprises ammonium bicarbonate and phosphate.

[0015] Preferably, the protease includes trypsin, Glu-C, and chymotrypsin, and the mass ratio of the protease to plasma protein is 1:10 to 1:100.

[0016] Preferably, in steps S2 and S4, the centrifugal force required for centrifugation is 9000-15000g, and the centrifugation time is 8min-15min.

[0017] Compared with the prior art, the present invention has the following beneficial effects: By utilizing the interaction between small molecule drugs and albumin to form a stable folded state, the structure and biophysical properties of proteins are affected, reducing the sensitivity of albumin to proteolytic enzymes and minimizing the impact of albumin on the identification of medium and low abundance proteins in plasma. This greatly improves the convenience of clinical sample analysis and significantly reduces costs. Attached Figure Description

[0018] Figure 1 This is a schematic flowchart of a method for pretreatment of plasma proteomics samples according to the present invention. Figure 2 This figure shows the number of proteins and peptides identified in three replicate experiments of the present invention using LTQ Orbitrap velos. Figure 3 This is a graph showing the correlation of protein intensity in three repeated experiments of this invention; Figure 4 This is a diagram showing the reproducibility of three repeated experiments in this invention. Figure 5 This is a graph showing the percentage of albumin peptide fragments in the spectrum of the present invention; Figure 6 A diagram showing the sequence and number of albumin peptides obtained using the sample pretreatment method described in this invention; Figure 7 The three most abundant albumin peptides obtained using the sample pretreatment method described in this invention; Figure 8 A graph showing the changes in FDA-approved biomarkers quantified in three replicate experiments. Detailed Implementation

[0019] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0020] Example 1 like Figure 1-8 As shown, a method for pretreatment of plasma proteomics samples involves adding a small molecule drug to the plasma sample and vortexing it for a period of time, simulating human body temperature and pH, to allow the small molecule drug to bind to albumin in the plasma and inhibit albumin enzymatic degradation. The treated plasma sample is then transferred to an ultrafiltration tube and centrifuged to remove small molecule metabolites and unbound small molecule drugs. Next, buffer and protease are added to the ultrafiltration tube to perform enzymatic digestion of the proteins. Finally, the external peptide fragments of the digested proteins are collected by centrifugation and analyzed by mass spectrometry.

[0021] It should be noted that, in the above scheme, the plasma sample is a clinically collected human plasma sample.

[0022] It should be noted that, in the above scheme, the small molecule drug is a drug that can bind to albumin, including but not limited to warfarin sodium, furosemide, dexamethasone and daptomycin. The above drugs can bind to albumin and have a high binding rate, which can improve the utilization rate of the drug.

[0023] It should be noted that in the above scheme, the concentration of the small molecule drug is 100mM-500mM to ensure a high binding rate when the small molecule drug binds to albumin.

[0024] It should be noted that in the above scheme, the human body temperature is 35-38℃, the pH value is 7.0-8.0, and the vortex time is 10-40min.

[0025] It should be noted that in the above scheme, the capacity of the ultrafiltration tube is 0.5-5 mL, and the specification of the ultrafiltration tube is 10 kD or 30 kD.

[0026] It should be noted that in the above scheme, the duration of the enzymatic digestion is 30 min to 2 h, which can limit the enzymatic digestion of peptides located outside the three-dimensional structure of the protein.

[0027] It should be noted that in the above scheme, the buffer solution includes ammonium bicarbonate and phosphate.

[0028] It should be noted that in the above scheme, the protease includes trypsin, Glu-C and chymotrypsin, and the mass ratio of the protease to plasma protein is 1:10 to 1:100.

[0029] It should be noted that in the above scheme, the centrifugal force required for centrifugation is 9000-15000g, and the centrifugation time is 8min-15min.

[0030] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for pretreatment of plasma proteomics samples, characterized in that, Includes the following steps: Step S1: Add the small molecule drug to the plasma sample. The small molecule drug interacts with albumin to form a stable folded state, thereby affecting the structure and biophysical properties of the protein, reducing the sensitivity of albumin to proteolytic enzymes, and swirling for a period of time to simulate human body temperature and pH value, so that the small molecule drug binds to albumin in the plasma and inhibits the enzymatic hydrolysis of albumin. Step S2: Transfer the plasma sample processed in step S1 to an ultrafiltration tube and centrifuge to remove small molecule metabolites and unbound small molecule drugs from the plasma. Step S3: Add buffer and protease to the ultrafiltration tube in step S2 to perform enzymatic digestion of the protein; Step S4: Centrifuge to collect the external peptides of the protein generated in step S4, and perform mass spectrometry analysis.

2. The method for pretreatment of plasma proteomics samples according to claim 1, characterized in that, In step S1, the plasma sample is a clinically collected human plasma sample.

3. The method for pretreatment of plasma proteomics samples according to claim 1, characterized in that, In step S1, the small molecule drug is a drug that can bind to albumin.

4. The method for pretreatment of plasma proteomics samples according to claim 1, characterized in that: In step S1, the concentration of the small molecule drug is 100mM-500mM.

5. The method for pretreatment of plasma proteomics samples according to claim 1, characterized in that, In step S1, the human body temperature is 35-38℃, the pH value is 7.0-8.0, and the vortex time is 10-40 min.

6. The method for pretreatment of plasma proteomics samples according to claim 1, characterized in that, In step S2, the capacity of the ultrafiltration tube is 0.5-5 mL.

7. The method for pretreatment of plasma proteomics samples according to claim 1, characterized in that, The enzyme digestion lasted for 30 minutes to 2 hours.

8. The method for pretreatment of plasma proteomics samples according to claim 1, characterized in that: The buffer solution comprises ammonium bicarbonate and phosphate.

9. A method for pretreatment of plasma proteomics samples according to claim 1, characterized in that, The proteases include trypsin, Glu-C, and chymotrypsin, and the mass ratio of the proteases to plasma proteins is 1:10 to 1:

100.

10. A method for pretreatment of plasma proteomics samples according to claim 1, characterized in that, In steps S2 and S4, the centrifugal force required for centrifugation is 9000-15000g, and the centrifugation time is 8min-15min.

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