WO2023185840A1

WO2023185840A1 - Mass spectrometry-based method for detecting medium- and low-abundance proteins in bodily fluid sample

Info

Publication number: WO2023185840A1
Application number: PCT/CN2023/084385
Authority: WO
Inventors: 朱奕颖
Original assignee: 清华大学
Priority date: 2022-04-01
Filing date: 2023-03-28
Publication date: 2023-10-05
Also published as: CN114593979A

Abstract

A mass spectrometry-based method for detecting medium- and low-abundance proteins of a sample, comprising: 1) removing only high-abundance proteins from a group of samples, realizing enrichment of low-abundance proteins, to be enhanced channel samples for subsequent detection; and 2) performing mass spectrometry analysis on the enhanced channel samples and on common channel samples using a high-throughput labeling method, to obtain quantitative results of each protein in bodily fluid. On the basis of the high-throughput labeling method, using an enhanced channel greatly increases mas spectrometry coverage of medium- and low-abundance proteins in blood, while retaining the high-abundance proteins in the samples.

Description

A method for detecting medium and low abundance proteins in body fluid samples based on mass spectrometry

Technical field

The present invention relates to the field of biological detection technology, and specifically to a method for detecting or assisting in the detection of medium and low abundance proteins in body fluid samples.

Background technique

Early diagnosis of disease is crucial for controlling disease development and prognosis. In actual clinical testing, non-invasive body fluid detection methods are used for early diagnosis of various diseases. Blood is the most commonly used body fluid test sample, and the proteins in it are commonly used diagnostic markers for diseases. Currently, more than one hundred in vitro diagnostic methods based on blood protein markers have been used clinically, including the diagnosis of infectious diseases, neurological diseases, cancer, diabetes, cardiovascular disease and prenatal diagnosis. In recent years, with the rapid development of mass spectrometry technology and the demand for medical precision, research related to the discovery of blood protein diagnostic markers through mass spectrometry detection has attracted more and more attention. TMT (Tandem Mass Tag) is the most commonly used high-throughput peptide labeling and quantification technology in the field of proteomics. It uses multiple isotope labels to label the amino groups of peptides in different samples, and can compare up to 18 samples on mass spectrometry at the same time. between protein expression levels, has been widely used in the field of blood proteomics.

However, blood proteome analysis has always faced difficulties. The fundamental reason is that the protein concentration in blood varies greatly, reaching 10 ¹² orders of magnitude, and it is difficult for mass spectrometry detection to cover samples with such a wide concentration range. The 14 high-abundance proteins in blood account for 95% of the total protein in blood, which greatly affects the detection of the remaining low-abundance proteins. Mass spectrometry needs to increase coverage and penetrate into low-abundance regions to detect proteins with diagnostic significance in the early stages of disease. In order to increase the protein detection rate, people use pretreatment methods to remove high-abundance proteins in blood samples to detect low-abundance proteins in blood. However, this method is cumbersome to operate, increases cost, and greatly limits the detection efficiency and application scope of blood proteome.

Patent US2021080469 discloses the use of tandem mass tags (TMT) isobaric labeling, a method developed for multiplex identification and quantification of proteins in single cell microsamples in a single LC-MS analysis/run. However, this method is only suitable for samples where the distribution of protein abundance in cells or tissue types is approximately normally distributed; when you want to use this method to detect proteins in body fluid samples such as blood, due to the protein abundance composition of blood There is a huge difference. Simply using this method cannot effectively detect low-abundance proteins in body fluids such as blood. Chua XY et al. disclosed the use of strong channel method in Tandem mass tag approach utilizing pervanadate BOOST channels delivers deeper quantitative characterization of the tyrosine phosphoproteome A method for detecting tyrosine phosphorylated proteins that are low in content and difficult to enrich. However, its method is mainly aimed at enriching and detecting a single type of (specifically phosphorylated) proteins in cells. If it is used to detect proteins in body fluid samples such as blood, it will not be able to enrich low-abundance proteins in the sample. It also cannot achieve complete detection of all types of low-abundance proteins. In other words, this method cannot meet the need to detect as many proteins as possible in body fluid samples such as blood.

invention disclosure

The purpose of the present invention is to provide a method for detecting low- and medium-abundance proteins in body fluid samples based on mass spectrometry.

The present invention provides a method for detecting medium and low abundance proteins in samples based on mass spectrometry, including:

1) Only high-abundance proteins in a group of samples are removed to enrich low-abundance proteins, which can be used as enhanced channel samples for subsequent detection;

2) Use high-throughput labeling method for mass spectrometry analysis of enhanced channel samples and ordinary channel samples to obtain quantitative results of various proteins in body fluids.

For the step 1

Proteins with a concentration higher than 1E ⁷ pg/mL in the blood are high-abundance proteins, and the rest are medium-low-abundance proteins. The high-abundance protein removal column of the method can remove 14 kinds of high-abundance proteins in blood, accounting for 95% of the total blood proteins, and the concentration of each protein ranges from 1E ⁷ to 5E ¹⁰ pg/mL. Some highly abundant proteins are listed in the table below:

The sample is a body fluid sample, and the body fluid sample can be a variety of body fluid types such as blood, saliva, cerebrospinal fluid, joint fluid or urine.

The sample is a blood sample, and the blood sample may be plasma or serum.

The sample is a blood sample, and the method for removing high-abundance proteins from blood in step 1) includes but is not limited to using a Top14 high-abundance protein removal spin column. Other high-abundance protein removal methods can also be used, and it is necessary to ensure that more than 95% of high-abundance proteins are removed. Other methods of improving low- and medium-abundance proteins can also be used, such as using nanoparticles to enrich low-abundance proteins, which must ensure that the total enrichment rate is greater than 20 times, or using a synthesized single protein or a mixture of several proteins as an enhanced channel sample. , it is necessary to ensure that the ratio of the protein concentration in the synthetic sample to the protein concentration in the blood sample is not higher than 100 times.

The invention also provides a low-abundance protein detection system, which includes a pre-processing device and a protein detection device; the pre-processing device is used to remove high-abundance proteins in the sample and enrich low-abundance proteins; the protein detection device Protein detection through high-throughput labeling methods.

The detection system can be implemented based on a liquid chromatography and mass spectrometry system.

The sample of the system is a liquid sample.

The liquid sample is a blood sample.

The pretreatment device includes a high-abundance protein removal medium-sized spin column; when the sample is a blood sample, the pretreatment device includes a Top14 high-abundance protein removal spin column.

The protein detection device includes a device for TMT high-throughput labeling method; the high-throughput labeling method includes but is not limited to TMT6plex, TMT10plex, TMT11plex, TMTpro16plex, TMTpro18plex, iTRAQ, etc.

The protein detection device also includes a mass spectrometer.

The application of the above-mentioned low-abundance protein detection system in detecting or assisting in the detection of low-abundance proteins in samples should also be within the protection scope of the present invention.

Description of drawings

Figure 1 is a schematic diagram of the principle of enhancing the channel to improve the coverage of low-abundance proteins.

Figure 2 shows a comparison of experimental technology roadmaps with and without the enhanced channel method.

Figure 3 is a schematic diagram of the experimental design in Example 1;

Figure 4 is a schematic diagram of the experimental design in Example 2.

Best way to implement your invention

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified. The room temperature mentioned in the following examples refers to 25°C.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

The present invention relates to a method for detecting low-abundance proteins in blood samples, and improves the mass spectrometry detection coverage of low-abundance proteins in blood through enhanced channels. This is the first time that the enhanced channel method is applied to blood proteomics. The innovation also includes the sample preparation of the enhanced channel using actual detection samples after removing high-abundance proteins.

The principle of the enhanced channel is as follows: When the TMT high-throughput labeling method performs quantitative analysis of proteins through mass spectrometry, the same peptide in different samples has the same mass-to-charge ratio after being labeled, and can be selected for secondary or even tertiary fragmentation at the same time. Fission produces reporter ions with different masses for quantitation. During mass spectrometry detection, usually only peptides with high relative abundance can be detected, while peptides with low relative abundance can be ignored. The newly added peptides in enhanced channel samples can greatly increase the relative abundance of low-abundance peptides in mixed samples, thus greatly increasing the probability of detection. Finally, the low-abundance peptides in other channels are reduced by reporter ions. Unified quantification (as shown in Figure 1).

This embodiment provides a method for detecting medium and low-abundance proteins in body fluid samples, as shown in Figure 2. The black part in Figure 2 is the ordinary classic TMT multi-channel blood proteomics step, and the gray part is the enhanced channel method. Unique steps include:

1) Take n blood samples according to the conventional method, take 1 μL each, and detect the protein concentration after urea lysis. Take a sample A of each sample, and perform reduction alkylation, trypsin digestion, desalting, spin drying, and reconstitution in sequence. , and then label it with TMT reagent to obtain the ordinary channel sample.

2) Take 1-2 μL of each of the n blood samples and combine them to obtain a mixed sample. Take 10 μL of the mixed sample for The high-abundance protein removal column removes high-abundance proteins. The mixed sample from which the high-abundance proteins are removed is lysed with urea, and sample B is also subjected to reductive alkylation, trypsin digestion, desalting, spin drying, and reconstitution, and then The enhanced channel sample was obtained by labeling it with TMT reagent.

3) Mix the normal channel sample with the enhanced channel sample, then desalt and analyze using LC-MS.

Among them, the sample amounts of A and B: If 14 high-abundance protein removal columns are used to remove about 95% of the proteins, the amount of protein in B is 1-5 times that of A, and the enrichment degree is 20-100 times. In other words, the ratio of the initial serum dosage for the ordinary channel and the enhanced channel is 1:20-1:100. That is, if the protein amount of the ordinary channel is 20 μg, the corresponding starting serum dosage is 0.2 μL, and the enhanced channel is 4-20 μL of combined serum. Remove the products of 14 highly abundant proteins.

Example 1

Through the method of the present invention, normal human serum samples are detected. The specific method is as follows:

1. Preparation of enhanced channel samples

Take 10 μL of serum samples from clinical healthy volunteers and use High-Select ^TM Top14 high-abundance protein removal medium spin column (Thermo Scientific #A36371) to remove high-abundance proteins. Add 10 μL of serum sample directly to the resin slurry in the column, mix by gently inverting at room temperature, and incubate for 10 minutes before centrifuging to collect the filtrate.

2. Preparation of samples for mass spectrometry detection

Take 1 μL of the same serum sample as step 1 and dissolve it in 200 μL of urea lysis solution (9M urea, 20mM 4-hydroxyethylpiperazinethanesulfonic acid). Take the sample filtrate in step 1 and add 2 mL of urea lysate. The protein concentration of all samples was determined using the Pierce BCA kit. Then, take an appropriate amount of dissolved serum protein solution, add dithiothreitol to 4.5mM and react at room temperature for 1 hour. Add iodoacetamide to 10mM and react at room temperature for half an hour in the dark. Add trypsin according to the mass ratio of trypsin:protein = 1:20 (w:w) and leave it at room temperature overnight. Add formic acid to the solution to 0.1%, use Stagetip C18 to desalt and set aside.

3. TMTpro reagent labeling of samples

Take 1 μg and 3 μg of the peptides obtained in step 2, and the peptides of the enhanced channel sample obtained in step 1 (approximately 3 μg of protein obtained after removing high-abundance proteins from 100 μg of mixed serum), and perform TMTpro according to the following method Reagent label:

Add 100 μL of 100 mM tetraethylammonium bromide buffer to the peptide sample, then add 30 μL of acetonitrile and the TMTpro reagent of the corresponding channel, mix and react at room temperature for 1 hour. The reaction was terminated by adding hydroxylamine to each sample to a concentration of 0.3%. As shown in Figure 3, mix 1 μg and 3 μg peptide samples, or mix samples from each channel and enhanced channel samples, and then use StagetipC18 to desalt to obtain samples without enhanced channels and with enhanced channels.

4.LC-MS detection

Spin the samples obtained in step 3 to dryness and then redissolve them with 0.1% formic acid solution. Detection was performed using UltiMate3000nanoUHPLC tandem Orbitrap Eclipse Tribrid mass spectrometer (Thermo Scientific). In the liquid phase, solution A is a 0.1% formic acid aqueous solution, and solution B is an 80% acetonitrile, 0.1% formic acid aqueous solution. The liquid phase gradient is B (80% acetonitrile, 0.1% formic acid) solution rising from 4% to 50% in 300 minutes, with a flow rate of 0.3 μL/min. The mass spectrometry method uses the Thermo TMT real-time search method, uses the ion trap to detect the secondary spectrum, and performs SPS3 Multinotch MS3 detection after real-time search. Mass spectrometry data were analyzed using Proteome Discoverer2.4 software (Thermo Scientific).

In the above method, in the enhancement channel, 100 μg of the serum protein enriched and purified product (in this experiment, an antibody spin column was used to remove 14 high-abundance proteins, and the purified protein amount was about 5 μg), was enzymatically hydrolyzed into enhanced Channel sample (the specific method is shown in step 2). The enhanced channel sample prepared in step 2 is TMT-labeled and mixed with the TMT-labeled ordinary channel sample, and the mixed sample is analyzed by LC-MS.

In this example, the multinotch MS3 method of the Thermo Orbitrap Eclipse mass spectrometer is used to analyze the peptide fragments. Finally, the Proteome Discoverer (PD) software is used to search the database and quantify the reported fragment ions. The results are shown in Table 1-3.

Table 1 shows the results of protein identification by the two methods. The quantitative protein number only includes proteins that were quantified in all channels. The enhanced channel method can significantly improve the qualitative and quantitative depth of blood proteome under the same conditions.

Table 1: Comparison of protein-to-protein qualitative and quantitative quantities with or without enhanced channels.

As shown in Table 2, three parallel experiments were performed on 1 μg and 3 μg serum proteins using the enhanced channel method and the non-enhanced channel method. The intermediate CV values of all jointly quantified proteins are within 20%. Comparing the methods with and without enhancement channels shows that there is no significant difference in the quantitative accuracy of the two methods.

Table 2. Comparison of repeatability of protein quantification with and without enhancement channel.

As shown in Table 3, the amount of serum protein in the sample is 3 μg and 1 μg, and the ratio is expected to be 3:1. Statistics of all jointly quantified protein ratios showed that the median protein ratio measured by the enhanced channel method was 3.6:1, while the median ratio measured by the non-enhanced channel method was 3.5:1. This demonstrates that the enhanced channel method maintains the accuracy of quantitative data.

Table 3. Comparison of relative quantification accuracy with and without enhancement channels.

Example 2

Through the method of the present invention, 7 cases of liver cancer patients and 7 cases of normal human serum samples were detected. The specific methods are as follows:

1. Preparation of enhanced channel samples

Serum samples from 14 patients and healthy people were mixed in equal proportions. Take 10 μL of the mixed serum sample and use High-Select ^TM Top14 high-abundance protein removal medium spin column (Thermo Scientific #A36371) to remove high-abundance proteins. Add 10 μL of serum sample directly to the resin slurry in the chromatographic column, mix by gently inverting at room temperature, incubate for 10 minutes and then centrifuge to collect the filtrate.

2. Preparation of samples for mass spectrometry detection

Dissolve 1 μL of each serum sample from 7 liver cancer patients and 7 normal people in 200 μL urea lysis solution (9M urea, 20mM 4-hydroxyethylpiperazinethanesulfonic acid). Take the enhanced channel sample filtrate in step 1 and add 2 mL of urea lysate. The protein concentration of all samples was determined using the Pierce BCA kit. Then, take an appropriate amount of dissolved serum protein solution, add dithiothreitol to 4.5mM and react at room temperature for 1 hour. Add iodoacetamide to 10mM and react at room temperature for half an hour in the dark. Add trypsin according to the mass ratio of trypsin:protein = 1:20 (w:w) and leave it at room temperature overnight. Add formic acid to the solution to 0.1%, use Stagetip C18 to desalt and set aside.

3. TMTpro reagent labeling of samples

Take the corresponding 14 serum samples and enhanced channel samples respectively, and label them with TMTpro reagent according to the following method:

Add 100 μL of 100 mM tetraethylammonium bromide buffer to the peptide sample, then add 30 μL of acetonitrile and the TMTpro reagent of the corresponding channel, and react at room temperature for 1 hour. The reaction was terminated by adding hydroxylamine to each sample to a concentration of 0.3%. As shown in Figure 4, mix the samples from each channel and the enhanced channel samples, and then use StagetipC18 to desalt to obtain samples without enhanced channels and with enhanced channels.

4.LC-MS detection

The samples obtained in step 3 were spin-dried and redissolved in 0.1% formic acid solution, and high pH reversed-phase HPLC pre-separation was performed, followed by desalting using stagetip C18. Detection was performed using UltiMate3000nanoUHPLC tandem Orbitrap Eclipse Tribrid mass spectrometer (Thermo Scientific). In the liquid phase, solution A is a 0.1% formic acid aqueous solution, and solution B is an 80% acetonitrile, 0.1% formic acid aqueous solution. The liquid phase gradient is solution B (80% acetonitrile, 0.1% formic acid) rising from 4% to 50% in 300 minutes, with a flow rate of 0.3 μL/min. The mass spectrometry method uses the Thermo TMT real-time search method, uses the ion trap to detect the secondary spectrum, and performs SPS3 Multinotch MS3 detection after real-time search. Mass spectrometry data were analyzed using Proteome Discoverer2.4 software (Thermo Scientific).

The results are shown in Table 4. In actual blood proteomics application research, the enhanced channel can significantly improve the protein detection rate.

Table 4: Comparison of blood protein detection results of 7 liver cancer patients and 7 normal subjects using different methods

As can be seen from Table 4, through the method of the present invention, 552 kinds of serum proteins can be detected (2.75 times that of ordinary detection), of which 210 kinds of serum proteins can be quantified (2.5 times that of ordinary detection); at the same time, patients with liver cancer The number of detected specific proteins compared with normal people is 28 (compared to only 18 in ordinary testing). This data shows that the method of the present invention can significantly increase the number of detected proteins and provide detection guarantee and convenient conditions for the research of medium and low abundance proteins.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

Industrial applications

Based on the high-throughput labeling method, the present invention uses enhanced channels to greatly improve the detection coverage of low-abundance protein spectra in blood on the premise of retaining high-abundance proteins in the sample. This method can help more researchers find blood protein markers that are truly related to diseases and assist in the diagnosis of various diseases.

By removing the high-abundance protein in the present invention, the sample is used as an enhanced channel sample to cooperate with the enhanced channel sample. This method can detect 552 kinds of serum proteins in blood samples (2.75 times that of ordinary detection), of which 210 kinds of serum proteins can be quantified (2.5 times that of ordinary detection); at the same time, the specificity of liver cancer patients and normal people The number of detected sexual proteins was 28 (only 18 in ordinary detection). This data shows that the method of the present invention can significantly increase the number of detected proteins and provide detection guarantee and convenient conditions for the research of medium and low abundance proteins.

Compared with the existing technology that uses enhanced channels to increase the detection of extremely low-abundance proteins in single-cell trace samples, the present invention is significantly different: the existing technology can detect absolute protein content lower than the lower limit of detection equipment such as mass spectrometry. Low-abundance proteins, however, when the sample contains both higher-abundance proteins and lower-abundance proteins (relatively low abundance), especially when the difference between the two is large, due to the particularity of mass spectrometry detection, this relatively low abundance Abundant proteins will not be detected by mass spectrometry under the influence of high-abundance protein data; however, this problem can be effectively solved by the method of the present invention, which is especially suitable for samples with huge differences in protein content, which greatly increases The types of proteins that can be detected in the sample. At the same time, low-abundance proteins in the present invention not only include the detection of proteins with lower absolute content, but also include proteins with not low absolute content but lower content compared to other proteins in the sample.

Claims

A method for detecting or assisting in the detection of low-abundance proteins in samples, which is characterized by including:

1) By preprocessing a group of samples to be tested, high-abundance proteins in the samples are removed, low-abundance proteins are enriched, and enhanced channel samples are obtained;

2) Mix the enhanced channel sample with the rest of the samples to be tested, and use high-throughput labeling method for mass spectrometry analysis to obtain quantitative results of the protein.
The method of claim 1, wherein the sample is a liquid sample.
The method of claim 2, wherein the sample is a blood sample or a urine sample or other body fluid sample.
The method of claim 3, wherein the sample is a blood sample, and in step 1), high-abundance proteins are removed through a high-abundance protein removal spin column.
The method of claim 1, wherein the high-throughput labeling method includes at least one of TMT6plex, TMT10plex, TMT11plex, TMTpro16plex, TMTpro18plex, and iTRAQ.
The application of a low-abundance protein detection system in detecting low-abundance proteins in samples. The low-abundance protein detection system includes a pretreatment device and a protein detection device; the pretreatment device is used to remove high-abundance proteins in the sample. detect proteins and enrich low-abundance proteins; the protein detection device detects proteins through a high-throughput labeling method.
The application according to claim 6, wherein the sample is a liquid sample; and the liquid sample is a body fluid sample such as a blood sample or a urine sample.
The application according to claim 6, characterized in that the sample is a blood sample, the pretreatment device includes a high-abundance protein removal medium-sized spin column; the protein detection device includes a high-throughput labeling method for TMT device.
The application according to any one of claims 7-8, characterized in that the protein detection device further includes a mass spectrometer.
A system for detecting low-abundance proteins, characterized in that the system includes a pre-treatment device and a protein detection device; the pre-treatment device is used to remove high-abundance proteins in a sample and enrich low-abundance proteins; The protein detection device detects proteins through a high-throughput labeling method.
The system according to claim 10, characterized in that the pretreatment device includes a high-density medium-sized spin column for protein removal; the protein detection device includes a device for TMT high-throughput labeling method; the protein detection device also includes a mass spectrometer.