High-throughput single-cell proteomics analysis method based on label combination and application of high-throughput single-cell proteomics analysis method

Abstract

The invention relates to a high-throughput single-cell proteomics analysis method based on label combination and application. The method is based on a tandem mass spectrometry labeling technology and comprises the step of adding a labeling reagent into a single-cell polypeptide sample, and the labeling reagent is composed of TMTpro 16plex and IBT-16plex. According to the analysis method disclosed by the invention, IBT-16plex and TMTpro 16 Plex markers are combined for use, and the IBT-16plex and TMTpro 16 Plex markers are applied to analysis and identification of single-cell proteomics, so that the flux of mass spectrum collection can be greatly improved, the cost is reduced, and meanwhile, the sensitivity is relatively high.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis, and in particular relates to a high-throughput single-cell proteomics analysis method based on labeling and application. Background Art

[0002] At present, the most important proteomics methods for improving throughput by labeling and quantification include: isotope tags for relative and absolute quantification (iTRAQ), tandem mass tag (TMT), stable-isotope labeling by amino acids in cell culture (SILAC), and label-free quantification.

[0003] iTRAQ technology is an in vitro peptide labeling technology developed by Applied Biosystems (ABI). iTRAQ technology uses a variety of isotope reagents to label the N-terminus or lysine side chain groups of protein peptides. After tandem analysis by a high-precision mass spectrometer, it can compare the protein expression levels of up to 10 samples at the same time. It is a high-throughput screening technology commonly used in quantitative proteomics in recent years; TMT technology is an in vitro peptide labeling technology developed by Thermo Scientific, USA. It has the same principle and advantages as iTRAQ technology, but has more labels than iTRAQ and can load more samples at one time. TMT technology uses 6, 10, 16 or 18 isotope labels to specifically label the amino groups of peptides, and then performs tandem mass spectrometry analysis to monitor the fragmented labels to achieve peptide quantification. One experiment can compare the relative content of proteins in up to 18 different samples at the same time; SILAC is an in vivo metabolic labeling technology that introduces stable isotopes through the use of 13C-glucose, 15NH or 3C labeled amino acids in the culture medium to label proteins in cells or small organisms and quantify them according to the mass spectrometry abundance ratio. Initially, the labeled amino acids used by SILAC were tritiated methionine and deuterated glycine. Currently, commonly used labeled amino acids include leucine, arginine, lysine, methionine, and tyrosine. Label-free protein quantification technology is a classic quantitative proteomics technology. This technology does not require expensive isotope (iTRAQ and TMT) labels as internal standards, but analyzes the quantitative changes in samples from different sources by comparing the number of mass spectrometry analyses or the intensity of mass spectrometry peaks. This method assumes that the frequency of peptide capture and detection in the mass spectrometry is positively correlated with its abundance in the mixture. Therefore, the count of protein detected by mass spectrometry reflects the abundance of the protein. The mass spectrometry detection count can be linked to the amount of protein through appropriate mathematical formulas, thereby quantifying the protein. Currently, the widely used labeling reagents for this technology are the TMT series, iTRAQ, and IBT series.

[0004] The existing TMT or IBT-based labeling technology can achieve up to 18 labels on Orbitrap mass spectrometers, and up to 9 labels on timsTOF SCP or timsTOF Pro mass spectrometers (due to the resolution of less than 50,000, the labeling reagents can only be used at intervals, and half of the labels can be used in total), and the throughput cannot be increased. SILAC has restrictions on sample types and is only applicable to living cultured cells. Tissue and body fluid samples cannot be analyzed, and the throughput is low. In addition, non-labeled quantitative technology that does not use labels has high requirements for experimental operation stability and low throughput.

[0005] There have been reports on the use of labeling to improve the throughput of single-cell proteomics. For example, in the reference Anal. Chem. 2023, 95, 5169−5175, three labeling reagents, TMTpro 18plex, IBT-16plex and TMTpro 11plex, were combined to analyze 45 samples at a time on the orbitrap mass spectrometry platform for a regular amount of proteome, but the regular amount of samples needed to be fractionated into 20 components, so the actual throughput improvement was only about 2.25 times. TMTpro 18plex and TMTpro 11plex have 11 shared channels in MS2 (i.e., all channels of TMTpro 11plex), and the m / z is exactly the same and cannot be distinguished, which leads to a decrease in quantitative accuracy. Moreover, this combination method still compares the samples labeled with the three labeling reagents, TMTpro 18plex, IBT-16plex and TMTpro 11plex, separately, without overall comparison, and without achieving true combination.

[0006] The combined use of TMT-16 and IBT-16 for single-cell proteomics has not been reported. Summary of the invention

[0007] In view of the above problems existing in the prior art, the present invention combines TMTpro 16plex and IBT-16plex for single-cell proteomics, and provides a high-throughput single-cell proteomics method and application based on label combination. The single-cell proteomics analysis method of the present invention can increase the detection throughput of single-cell samples by up to 30 times.

[0008] The technical solution of the present invention is: A high-throughput single-cell proteomics analysis method based on labeling combination, the method is based on tandem mass spectrometry labeling technology, and includes the step of adding a labeling reagent to a single-cell polypeptide sample, wherein the labeling reagent consists of TMTpro 16plex and IBT-16plex.

[0009] The structural formula of TMTpro is as follows:

[0010] The structural formula of IBT-16plex is as follows:

[0011] The TMTpro 16plex isotope labeling reagent has a longer linker group and a reporter group in its structure. The reporter group contains 9 stable C13 and N15 isotopes, which can support more channels.

[0012] IBT-16plex is a recently developed domestic labeling reagent. Its reporter ion mass is between 114 and 122. Compared with the TMT series reagents (126-135), its reporter ion is completely different from TMTpro 16plex, and there is no shared channel between the two. In this way, interference from reporter ions of different labeling reagents can be avoided.

[0013] The present application scheme uses TMTpro 16plex and IBT-16plex labeling reagents in combination to analyze multiple single-cell samples at a time, such as 16 single-cell samples in one embodiment. The identification amount of a single labeling channel can reach about 3,000 proteins, which greatly improves the detection throughput and also has high technical sensitivity.

[0014] Preferably, the analysis method of the present invention comprises the following steps: 1) Extract single-cell protein and obtain single-cell peptide samples; this step does not have too many restrictions. Depending on the cell type, you can add lysis buffer, enzymatic solution, etc. to lyse the cells and extract peptides according to the conventional extraction method.

[0015] 2) adding a labeling reagent to the single cell polypeptide sample, mixing, reacting, and preparing a mass spectrometry loading sample; Preferably, in step 2), the labeling reagents with different labels are first added to a single reaction container, mixed with the polypeptide sample, and reacted. After the reaction is terminated, the samples in each single reaction container are centrifuged and mixed to obtain a mass spectrometry loading sample. In this way, multiple cells can be analyzed at one time, thereby improving the detection throughput.

[0016] Preferably, in step 2), the protein concentration in each single reaction container is 0-5 ng / µL.

[0017] Preferably, in step 2), the concentration of the labeling reagent of a single label in each single reaction container is 0.5-5 µg / µL.

[0018] 3) Load the sample for mass spectrometry analysis to obtain qualitative data on the number of proteins in each labeled channel. There are no special requirements for the type of mass spectrometry, and the mass spectrometry injection measurement method can be performed according to conventional operations.

[0019] Further preferably, the analysis method of the present invention further comprises the step: 4) using a normalization method to process the obtained protein number qualitative data of each labeled channel. After normalization, the sensitivity difference between TMTpro 16plex and IBT-16plex reagents is leveled, which is more conducive to overall combined analysis.

[0020] Preferably, the normalization method may be to normalize the conversion coefficient of each protein construction, or to use an additional reference channel correction for normalization. Specifically, The conversion coefficient constructed for each protein was normalized; that is, according to the quantitative search results of the IBT group and the TMT group, the commonly identified protein expression matrix was merged, the quantitative data of each labeling channel of different groups were obtained in the merged protein matrix, the missing values ​​were filled with the minimum value, and the mean of the quantitative results of different labeling channels of different proteins in the quantitative matrix was obtained to form a conversion coefficient between the two TMT-IBT groups unique to different proteins, and the protein conversion coefficient was used to convert the quantitative results of each labeling channel of other samples.

[0021] An additional reference channel was used for normalization, that is, an additional channel was taken out from each group of IBT and TMT as a reference channel, and the same amount of standard was added to the reference channel, that is, the amount of various proteins was fixed, so other channels and multiple batches of samples could be calibrated using the reference channel to obtain relative quantitative results. After correction, the overall ratio also conforms to the quantitative relationship of the sample itself; Preferably, the reference channel is added with 1-3x the amount of standard of the single cell sample. The reference channel can be any channel, usually the first channel, but can also be other channels.

[0022] In the scheme of the present invention, the mass spectrometer can be a high-resolution mass spectrometer such as a timsTOF SCP mass spectrometer, a timsTOF HT mass spectrometer, a timsTOF Ultra mass spectrometer, an orbitrap mass spectrometer, or an Astral mass spectrometer.

[0023] Another aspect of the present invention also provides the application of the high-throughput single-cell proteomics analysis method based on labeling combination in the proteomics analysis and identification of a single cell or a small number of cells.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The high-throughput single-cell proteomics analysis method based on labeling combination of the present invention has the advantages of high throughput and high sensitivity, far exceeding the existing level in terms of flux, and is significantly better than the currently published higher-throughput labeling methods in terms of detection depth, and realizes the overall comparison between different labeling reagents for the first time. Combined with the labeling technology, 16 labeling channels can be used at a time on the timsTOFSCP, and 32 labeling channels can be used at a time on other higher-resolution mass spectrometers. The identification amount of a single labeling channel can reach about 3,000 proteins. Compared with label-free single-cell proteomics quantitative technology, the technology of the present invention greatly improves the detection flux of mass spectrometry and saves time cost. Compared with other labeling quantitative single-cell proteomics technologies, the technology of the present invention has high sensitivity and has obvious advantages in the identification depth of proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of the analysis and evaluation of the simulated single cell samples containing 50 times carriers labeled with IBT-16plex and TMTpro 16plex in Group A of Example 1; (A) labeling efficiency, (B) qualitative number of proteins in each labeling channel, (C) quantitative accuracy of the two labeling reagents, D) comparison before and after normalization using method 1, (E) comparison before and after normalization using method 2; Figure 2 The results of the analysis and evaluation of the simulated single cell samples containing 50 times carriers labeled with IBT-16plex and TMTpro 16plex in Group B of Example 1; (A) labeling efficiency, (B) qualitative number of proteins in each labeling channel, (C) quantitative accuracy of the two labeling reagents, (D) comparison before and after normalization using method 1, (E) comparison before and after normalization using method 2; Figure 3 The results of the analysis and evaluation of the simulated single cell sample containing 50 times carrier labeled by IBT-16plex in Group D of Example 1; (A) labeling efficiency, (B) qualitative number of proteins in each labeling channel, (C) quantitative accuracy of IBT-16plex labeling reagent; Figure 4 The number of protein identifications in each labeling channel in Example 2 when 20 293T single cells were labeled with IBT-16plex and TMTpro 16 Plex, and the sample was loaded with a single needle; Figure 5 The number of proteins in each labeled channel of the single-labeled 293T single cell in Example 2 is qualitative; wherein (A) is the number of proteins in each labeled channel of the TMT-labeled 293T single cell, and (B) is the number of proteins in each labeled channel of the IBT-labeled Hela single cell. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0028] Source of reagents: TMTpro 16Plex: Thermo Fisher Scientific; IBT-16plex: Nanjing Plexus Technology Co., Ltd.

[0029] Unless otherwise specified, other reagents, materials, and equipment used in the examples of the present invention are all commercially available; unless otherwise specified, the test methods are all conventional test methods in the art.

[0030] Example 1 Quantitative comparison experiment of simulated single cell samples (1) Preparation of labeling reagents.

[0031] Both IBT-16plex and TMTpro 16Plex labeling reagents are 0.5 mg / tube. After taking out the labeling reagent from the -20 ℃ refrigerator, place it at room temperature for a while, centrifuge it at 20000 r for 2 minutes, and let all the reagents concentrate at the bottom of the tube. Then slowly open the lid, quickly add 100 µL of anhydrous acetonitrile, and mix well. After centrifugation, dispense it into low-absorption tubes, 10 µL per tube.

[0032] Labeling reagent group: IBT_A group: the eight labels 114, 115C, 116C, 117C, 118C, 119C, 120C, and 121C in the IBT-16plex are divided into the IBT_A group; IBT_B group: the eight labels 115N, 116N, 117N, 118N, 119N, 120N, 121N, and 122 in the IBT-16plex are divided into the IBT_B group; TMT_A group: 8 tags 126, 127C, 128C, 129C, 130C, 131C, 132, and 133C in TMTpro 16Plex were divided into TMT_A group; TMT_B group: 127N, 128N, 129N, 130N, 131N, 132N, 133N, and 134N in TMTpro 16Plex are divided into TMT_B group.

[0033] (2) Prepare Hela digest solutions of different concentrations as standards.

[0034] (a) Dilute HeLa digest at a concentration of 50 ng / µL to a concentration of 10 ng / µL.

[0035] (b) Hela digest at a concentration of 10 ng / µL was diluted to a concentration of 1 ng / µL.

[0036] (c) Hela digest at a concentration of 1 ng / µL was diluted to 0.5 ng / µL, 0.3 ng / µL, 0.2 ng / µL, and 0.1 ng / µL.

[0037] (3) Conduct labeling experiments.

[0038] The experiment was set up with two groups, A and B. Each group of experiments was repeated three times, and each group prepared 16 low-adsorption centrifuge tubes and numbered them. The experimental steps are shown in Table 1.

[0039] Table 1 Labeling experiment using IBT-16plex and TMTpro 16Plex labeling reagents

[0040] (4) Conduct a labeling control experiment.

[0041] The experiment was set up with two groups of IBT-16plex single labeling experiments, C and D. Each group of experiments was repeated three times, and each group prepared 8 low-adsorption centrifuge tubes and numbered them. The experimental steps are as follows: Table 2 IBT-16plex single labeling reagent combined labeling experiment

[0042] (5) Normalization processing.

[0043] In this embodiment, two methods are used to normalize samples labeled with a combined labeling reagent.

[0044] Normalization method 1: Normalize the conversion coefficients of each protein construct.

[0045] This normalization method is based on the combination of IBT-TMT multiple samples. According to the quantitative search results of the IBT group and the TMT group, the commonly identified protein expression matrix is ​​obtained by merging, and the quantitative data of each labeling channel of different groups are obtained in the merged protein matrix. The missing values ​​are filled with the minimum value, and the mean of the quantitative results of different proteins in the quantitative matrix are obtained. The conversion coefficient between the two TMT-iBT groups unique to different proteins is constructed, and the quantitative results of each labeling channel of other samples are converted using the protein conversion coefficient.

[0046] Normalization method 2: using additional reference channel correction.

[0047] In each group of IBT and TMT, an additional channel is taken out (usually the first channel is used, and 1-3x the amount of single-cell sample standard is added) as the reference channel. The same amount of standard is added to the reference channel. The amount of various proteins is fixed, so other channels and multiple batches of samples can be calibrated using the reference channel to obtain relative quantitative results. After calibration, the overall ratio conforms to the quantitative relationship of the sample itself.

[0048] Experimental Results The experimental results are as follows Figure 1-3 As shown, Figure 1 Analysis and evaluation of simulated single cell samples containing 50 times carrier labeled by IBT-16plex and TMTpro 16plex in group A. (A) Labeling efficiency. (B) Qualitative number of proteins in each labeled channel. (C) Box plot of the quantitative accuracy of the two labeling reagents. (D) Comparison of method 1 before and after normalization. (E) Comparison of method 2 before and after normalization.

[0049] Figure 2 Analysis and evaluation of simulated single cell samples containing 50x carriers labeled with IBT-16plex and TMTpro 16plex in group B. (A) Labeling efficiency. (B) Qualitative number of proteins in each labeled channel. (C) Box plot of the quantitative accuracy of the two labeling reagents. (D) Comparison of method 1 before and after normalization. (E) Comparison of method 2 before and after normalization.

[0050] Figure 3 Analysis and evaluation of simulated single cell samples containing 50x carriers labeled with IBT-16plex in group D. (A) Labeling efficiency. (B) Qualitative number of proteins in each labeled channel. (C) Box plot of the quantitative accuracy of IBT-16plex labeling reagent.

[0051] It can be seen that the analysis effect of the combined use of IBT-16plex and TMTpro 16Plex labeling is better than that of a single labeling, in which the labeling efficiency is slightly higher than that of a single labeling. The number of protein identifications obtained by the two labeling methods is similar, and the number of protein identifications is slightly higher when labeling with a single labeling reagent. However, the throughput can be increased by about one-fold when the labels are used in combination, which can greatly improve the mass spectrometry detection throughput. Example 2 Single Cell Proteomics Application Experiment The labeling and quantitative method of the present invention was applied to the actual single cell determination. We selected 293T cells. The specific experimental method is shown in Example 1.

[0052] The number of protein identifications per channel for a single cell is Figure 4 shown. Figure 420 293T single cells were labeled with IBT-16plex and TMTpro 16Plex labeling reagents, and the number of protein identifications in each labeling channel was plotted on a single needle. The figure shows the results of three replicates.

[0053] For comparison, we also used a single labeling reagent to label single cells, such as using each channel in the TMTpro 16Plex-B group to label 293T single cells, and using each channel in the IBT-16plex-B group to label Hela single cells. The results are as follows: Figure 5 As shown, (A) is the qualitative number of proteins in each labeled channel for TMT-labeled 293T single cells. (B) is the qualitative number of proteins in each labeled channel for IBT-labeled Hela single cells.

[0054] By comparison, it can be seen that when applied to actual cell testing, the number of protein identifications obtained by the two labeling methods is similar. The number of protein identifications is slightly higher when labeled with a single labeling reagent, but the mass spectrometry detection throughput can be increased by about one-fold when the labels are used in combination.

[0055] In the above embodiment, the combined mass spectrometry analysis is based on the timsTOF SCP mass spectrometer. Due to the limitation of the secondary spectrum resolution, only half of the throughput can be used for a single sample loading. If the TMTpro 16Plex and IBT16-Plex labeling combination method of the present invention is analyzed using an orbitrap mass spectrometer, Astral, timsTOF HT, timsTOF Ultra and other higher resolution mass spectrometers, the detection throughput of single cell samples can be increased by up to 30 times.

[0056] In summary, the feasibility of the combined labeling of IBT-16plex and TMTpro 16 Plex in the present application scheme is demonstrated. Applying it to the analysis and identification of single-cell proteomics can greatly improve efficiency and reduce costs, while also having high sensitivity.

[0057] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0058] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A high-throughput single-cell proteomics analysis method based on labeling, the method is based on tandem mass spectrometry labeling technology, including the step of adding a labeling reagent to a single-cell polypeptide sample, characterized in that: The labeling reagent consists of TMTpro16plex and IBT-16plex.

2. The method according to claim 1, characterized in that The steps include: 1) Prepare single cell samples into peptide samples; 2) adding a labeling reagent to the single cell polypeptide sample, mixing, reacting, and preparing a mass spectrometry loading sample; 3) Load the sample for mass spectrometry analysis to obtain qualitative and quantitative data of proteins in each labeled channel.

3. The method according to claim 2, characterized in that: In step 2), the labeling reagents with different labels are first added to single reaction containers, mixed with the polypeptide samples, and reacted. After the reaction is terminated, the samples in each single reaction container are centrifuged and mixed to obtain mass spectrometry loading samples.

4. The method according to claim 3, characterized in that In step 2), the protein concentration in each single reaction vessel is 0-5 ng / µL.

5. The method according to claim 3, characterized in that: In the step 2), the concentration of the labeling reagent of a single tag in each single reaction container is 0.5-5 µg / µL.

6. The method according to any one of claims 2 to 5, characterized in that: Also includes the steps: 4) The obtained qualitative data of protein number of each labeling channel are processed by a normalization method.

7. The method according to claim 6, characterized in that The normalization method is: The conversion factor was normalized for each protein construct; That is, according to the quantitative search results of the IBT group and the TMT group, the protein expression matrix of the commonly identified proteins was obtained by merging, the quantitative data of each labeling channel of different groups were obtained in the merged protein matrix, the missing values ​​were filled with the minimum value, and the mean of the quantitative results of different labeling channels of different proteins in the quantitative matrix was obtained to form a conversion coefficient between the two TMT-IBT groups unique to different proteins, and the protein conversion coefficient was used to convert the quantitative results of each labeling channel of other samples; Alternatively, an additional reference channel calibration is used for normalization, that is, an additional channel is taken out from each group of IBT and TMT as a reference channel, the same amount of standard is added to the reference channel, and the reference channel is used for calibration to obtain relative quantitative results of other channels and / or channels of multiple batches.

8. The method according to claim 7, characterized in that The reference channel is any one of the channels, with 1-3x the amount of single cell sample standard added.

9. The method according to claim 1, characterized in that: The mass spectrum is a high-resolution mass spectrum.

10. Use of the high-throughput single-cell proteomics analysis method based on labeling combination according to any one of claims 1 to 9 in proteomics analysis and identification of single cells or other trace samples.

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