A high-throughput single-cell proteomics analysis method based on marker combination and application

Abstract

The application relates to a high-throughput single-cell proteomics analysis method based on a combination of markers and application. The method is based on a tandem mass spectrometry labeling technology, and comprises the step of adding a labeling reagent to a single-cell polypeptide sample, wherein the labeling reagent is composed of TMTpro 16plex and IBT-16plex. The analysis method of the application combines IBT-16plex and TMTpro 16 Plex labeling, applies the same to single-cell proteomics analysis and identification, can greatly improve the throughput of mass spectrometry acquisition, reduce the cost, and has high sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a high-throughput single-cell proteomics analysis method and its application based on label-coupled assays. Background Technology

[0002] The most important labeling and quantification proteomics methods to improve throughput at present include: isobaric tags for relative and absolute quantification (iTRAQ), tandem mass tag (TMT), stable isotope labelling by amino acids in cell culture (SILAC), and label-free quantification techniques.

[0003] iTRAQ technology is an in vitro peptide labeling technique developed by Applied Biosystems (ABI). iTRAQ uses multiple isotopic reagents to label the N-terminus or lysine side chain groups of protein peptides, followed by tandem mass spectrometry analysis. It can simultaneously compare protein expression levels among up to 10 samples, making it a commonly used high-throughput screening technique in quantitative proteomics in recent years. TMT technology is an in vitro peptide labeling technique developed by Thermo Scientific. It shares the same principle and advantages as iTRAQ, but offers more tags, allowing for more samples to be processed simultaneously. TMT uses 6, 10, 16, or 18 isotopic tags to specifically label the amino groups of peptides, followed by tandem mass spectrometry analysis to monitor fragmented tags and quantify peptides. A single experiment can simultaneously compare the relative protein content in up to 18 different samples. SILAC is an in vivo metabolic labeling technique that introduces stable isotopes into a culture medium labeled with 13C-glucose, 15NH, or 3C amino acids, labeling proteins in cells or small organisms and quantifying them based on mass spectrometry abundance ratios. Initially, SILAC used tritium-methionine and deuterated glycine as labeled amino acids. Currently, commonly used labeled amino acids include leucine, arginine, lysine, methionine, and tyrosine. Label-free protein quantification is a classic quantitative proteomics technique. This technique does not require expensive isotope tags (iTRAQ and TMT) as internal standards. Instead, it analyzes the changes in the quantity of proteins 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 mass spectrometry is positively correlated with its abundance in the mixture. Therefore, the protein count detected by mass spectrometry reflects the protein abundance. Through appropriate mathematical formulas, the mass spectrometry detection count can be linked to the amount of protein, thereby quantifying the protein. Currently, the widely used labeling reagents for this technique are the TMT series, iTRAQ, and IBT series.

[0004] Existing TMT or IBT-based labeling technologies can achieve a maximum of 18 labels on Orbitrap-type mass spectrometers and a maximum of 9 labels on timsTOF SCP or timsTOF Pro mass spectrometers (due to resolutions below 50,000, labeling reagents can only be used intermittently, with a total of half the labels usable), with throughput not being able to be further increased. SILAC has limitations on sample types, only suitable for in vivo cultured cells; tissue and body fluid samples cannot be analyzed, and throughput is low. In addition, label-free non-standard quantitative techniques require high experimental stability and have low throughput.

[0005] There have been reports of using labeling conjugations to improve the throughput of single-cell proteomics. For example, in Anal. Chem. 2023, 95, 5169−5175, three labeling reagents, TMTpro 18plex, IBT-16plex, and TMTpro 11plex, were conjugated. This allowed for the analysis of 45 samples in a single run on the Orbitrap mass spectrometry platform for routine proteomics. However, since routine samples need to be fractionated into 20 fractions, the actual throughput increase is only about 2.25 times. TMTpro 18plex and TMTpro 11plex share 11 channels in MS2 (i.e., all channels of TMTpro 11plex), and their m / z values ​​are identical, making them indistinguishable and leading to decreased quantitative accuracy. Moreover, this conjugation method still compares samples labeled with the three reagents separately, without overall comparison, thus failing to achieve true conjugation.

[0006] There have been no reports of combining TMT-16 and IBT-16 for single-cell proteomics. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, this invention combines TMTpro 16plex and IBT-16plex for single-cell proteomics, providing a high-throughput single-cell proteomics method and application based on label-coupled techniques. The single-cell proteomics analysis method of this invention can increase the detection throughput of single-cell samples by up to 30 times.

[0008] The technical solution of this invention is as follows:

[0009] A high-throughput single-cell proteomics analysis method based on labeling is disclosed. The method is based on tandem mass spectrometry labeling technology and includes the step of adding labeling reagents to a single-cell peptide sample. The labeling reagents consist of TMTpro 16plex and IBT-16plex.

[0010] The structural formula of TMTpro is as follows:

[0011]

[0012] The structural formula of IBT-16plex is as follows:

[0013]

[0014] 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.

[0015] IBT-16plex is a recently developed domestically produced 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 that of TMTpro 16plex, and there is no shared channel between the two. Thus, interference from reporter ions from different labeling reagents can be avoided.

[0016] This application's solution uses TMTpro 16plex and IBT-16plex labeling reagents in combination, which can analyze multiple single-cell samples in a single run. For example, in one embodiment, 16 single-cell samples can be analyzed, and the identification capacity of a single labeling channel can reach about 3,000 proteins, which greatly improves the detection throughput and also has high technical sensitivity.

[0017] Preferably, the analysis method of the present invention includes the following steps:

[0018] 1) Extract single-cell proteins to obtain single-cell peptide samples; there are not many restrictions on this step. Depending on the cell type, the peptides can be extracted by adding lysis buffer, enzyme digestion solution, etc., according to the conventional extraction method.

[0019] 2) Add labeling reagents to the single-cell polypeptide sample, mix, react, and prepare a mass spectrometry sample;

[0020] Preferably, in step 2), the labeling reagents with different labels are first added to separate reaction containers, mixed with the peptide sample, and reacted. After the reaction is terminated, the samples in each individual reaction container are centrifuged and then mixed to obtain the mass spectrometry sample. This allows for the analysis of multiple cells at once, increasing the detection throughput.

[0021] Preferably, in step 2), the protein concentration in each individual reaction vessel is 0-5 ng / µL.

[0022] Preferably, in step 2), the concentration of the labeling reagent of a single label in each individual reaction vessel is 0.5-5 µg / µL.

[0023] 3) Sample loading and mass spectrometry analysis to obtain qualitative data on the protein count in each labeled channel. There are no special requirements for the type of mass spectrometer; the mass spectrometry sample injection and measurement method can be followed as usual.

[0024] Further preferably, the analytical method of the present invention further includes the step: 4) processing the qualitative data of protein counts in each labeled channel obtained by normalization. After normalization, the sensitivity difference between TMTpro 16plex and IBT-16plex reagents is leveled out, which is more conducive to overall coupled analysis.

[0025] Preferably, the normalization method can be to normalize the conversion coefficient of each protein construction, or it can be to normalize using an additional reference channel correction. Specifically,

[0026] For each protein, a conversion coefficient was constructed and normalized. Specifically, based on the quantitative search results of the IBT and TMT groups, a commonly identified protein expression matrix was obtained. Quantitative data for each label channel of different groups were obtained from the merged protein matrix. Missing values ​​were filled using the minimum value, and the mean of the quantitative results for different label channels of different proteins in the quantitative matrix was obtained. This constituted a conversion coefficient between the TMT-IBT groups unique to different proteins. The protein conversion coefficient was then used to convert the quantitative results of each label channel of other samples.

[0027] Normalization was performed using an additional reference channel. Specifically, an extra channel was selected as a reference channel for each of the IBT and TMT groups. The same amount of standard was added to the reference channel, ensuring a fixed amount of each protein. Therefore, other channels and multiple batches of samples could be corrected using the reference channel to obtain relative quantification results. The overall proportions after correction also conformed to the quantification relationships inherent in the samples themselves.

[0028] Preferably, the reference channel contains 1-3 times the amount of a single-cell sample of standard. The reference channel can be any channel, typically the first channel, but it can also be any other channel.

[0029] In this invention, the mass spectrometer can be a high-resolution mass spectrometer such as timsTOF SCP, timsTOF HT, timsTOF FULTRA, orbitrap, or Astral.

[0030] In another aspect, the present invention also provides the application of the aforementioned label-based high-throughput single-cell proteomics analysis method in proteomics analysis and identification of single cells or a small number of cells.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention presents a high-throughput single-cell proteomics analysis method based on label-coupled techniques, offering advantages in both high throughput and high sensitivity. Its throughput far surpasses existing levels, and its detection depth is significantly superior to currently published high-throughput label-based methods. Furthermore, it achieves, for the first time, a comprehensive comparison between different labeling reagents. By combining labeling techniques, 16 labeling channels can be used in a single run on timsTOFSCP, and 32 labeling channels can be used in a single run on other higher-resolution mass spectrometers. A single labeling channel can identify approximately 3000 proteins. Compared to label-free single-cell proteomics quantification techniques, this invention significantly improves the detection throughput of mass spectrometry and saves time. Compared to other label-based quantitative single-cell proteomics techniques, this invention offers high sensitivity and a significant advantage in protein identification depth. Attached Figure Description

[0033] Figure 1 The results of the analysis and evaluation of simulated single-cell samples containing 50-fold carrier labeled with IBT-16plex and TMTpro 16Plex in Group A of Example 1 are as follows: (A) Labeling efficiency, (B) Qualitative number of proteins in each labeled channel, (C) Quantitative accuracy of the two labeling reagents, (D) Comparison before and after normalization using Method 1, and (E) Comparison before and after normalization using Method 2.

[0034] Figure 2 The results of the analysis and evaluation of simulated single-cell samples containing 50-fold carrier labeled with IBT-16plex and TMTpro 16Plex in Group B of Example 1 are as follows: (A) Labeling efficiency, (B) Qualitative number of proteins in each labeled channel, (C) Quantitative accuracy of the two labeling reagents, (D) Comparison before and after normalization using Method 1, and (E) Comparison before and after normalization using Method 2.

[0035] Figure 3 The results of the analysis and evaluation of simulated single-cell samples labeled with IBT-16plex and containing 50-fold carrier in Group D of Example 1 are as follows: (A) Labeling efficiency, (B) Qualitative number of proteins in each labeled channel, and (C) Quantitative accuracy of IBT-16plex labeling reagent.

[0036] Figure 4 In Example 2, IBT-16plex and TMTpro 16 Plex were used to label 20 293T single cells, with single-needle loading, and the number of proteins identified in each labeling channel was recorded.

[0037] Figure 5The qualitative count of protein count per labeled channel in a single-labeled 293T single cell in Example 2 is given by (A) and (B) is given by (B) and (C) is given by (D) and (D) is given by (IBT) and (C) is given by (D) and (D) is given by (D ... Detailed Implementation

[0038] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0040] Reagent source:

[0041] TMTpro 16Plex: Thermo Fisher Scientific;

[0042] IBT-16plex: Nanjing Pulijian Company.

[0043] Unless otherwise specified, all other reagents, materials, and equipment used in the embodiments of this invention are commercially available; unless otherwise specified, the test methods are conventional test methods in the art.

[0044] Example 1: Quantitative Comparison Experiment of Simulated Single-Cell Samples

[0045] (1) Preparation of labeling reagents.

[0046] Both IBT-16plex and TMTpro 16Plex labeling reagents were administered at 0.5 mg / tube. After removing the labeling reagents from the -20 °C freezer, allow them to stand at room temperature for a short time, then centrifuge at 20,000 r for 2 minutes to concentrate the reagent at the bottom of the tube. Then, slowly open the cap and quickly add 100 µL of anhydrous acetonitrile, mixing thoroughly. After centrifugation, aliquot the solution into low-adsorption tubes, 10 µL per tube.

[0047] Labeling reagent grouping:

[0048] Group IBT_A: The eight tags 114, 115C, 116C, 117C, 118C, 119C, 120C, and 121C in the IBT-16plex are divided into Group IBT_A.

[0049] Group IBT_B: The eight tags 115N, 116N, 117N, 118N, 119N, 120N, 121N, and 122 in the IBT-16plex are divided into Group IBT_B.

[0050] TMT_A Group: The eight tags 126, 127C, 128C, 129C, 130C, 131C, 132, and 133C in TMTpro 16Plex are divided into TMT_A group;

[0051] TMT_B Group: The 8 tags 127N, 128N, 129N, 130N, 131N, 132N, 133N, and 134N in TMTpro 16Plex are divided into TMT_B group.

[0052] (2) Prepare Hela digest solutions of different concentrations as standards.

[0053] (a) Dilute Hela digest at a concentration of 50 ng / µL to a concentration of 10 ng / µL.

[0054] (b) Dilute Hela digest at a concentration of 10 ng / µL to a concentration of 1 ng / µL.

[0055] (c) HeLa digest at a concentration of 1 ng / µL was diluted to concentrations of 0.5 ng / µL, 0.3 ng / µL, 0.2 ng / µL, and 0.1 ng / µL, respectively.

[0056] (3) Conduct a labeling experiment.

[0057] The experiment was set up in two groups, A and B. Each group was repeated three times, and each group prepared 16 low-adsorption centrifuge tubes and numbered them. The experimental steps are shown in Table 1 below.

[0058] Table 1. Labeling experiments using IBT-16plex and TMTpro 16Plex labeling reagents in combination.

[0059]

[0060] (4) Conduct a label control experiment.

[0061] The experiment was set up in two groups, C and D, for single-labeling of IBT-16plex. Each group was repeated three times, and each group prepared eight low-adsorption centrifuge tubes, which were then numbered. The experimental procedures are shown in the table below:

[0062] Table 2. Labeling experiments using IBT-16plex single labeling reagents in combination.

[0063]

[0064] (5) Normalization process.

[0065] In this embodiment, two methods were used to normalize the samples labeled with the combined labeling reagents.

[0066] Normalization method 1: Normalize the conversion coefficients for each protein construction.

[0067] This normalization method is based on the multi-sample combination of IBT-TMT. According to the quantitative search results of the IBT group and the TMT group, the commonly identified protein expression matrix is ​​obtained by merging. The quantitative data of each label channel of different groups are obtained in the merged protein matrix. The missing values ​​are filled with the minimum value. The mean value of the quantitative results of different label channels of different proteins in the quantitative matrix is ​​obtained, which constitutes the conversion coefficient between the two TMT-iBT groups for different proteins. The protein conversion coefficient is used to convert the quantitative results of each label channel of other samples.

[0068] Normalization method 2: Use an additional reference channel for correction.

[0069] In each group of IBT and TMT, an additional channel is selected (usually the first channel is used, with 1-3 times the amount of single-cell sample standard added) as a reference channel. The same amount of standard is added to the reference channel, and the amounts of various proteins are fixed. Therefore, other channels and multiple batches of samples can be corrected using the reference channel to obtain relative quantification results. After correction, the overall proportions conform to the quantification relationship of the sample itself.

[0070] Experimental results

[0071] Experimental results are as follows Figure 1-3 As shown, where Figure 1 Evaluation of simulated single-cell samples labeled with IBT-16plex and TMTpro 16Plex in group A, containing 50-fold carriers. (A) Labeling efficiency. (B) Qualitative protein count for each labeled channel. (C) Box plot of quantitative accuracy for the two labeling reagents. (D) Comparison of method 1 before and after normalization. (E) Comparison of method 2 before and after normalization.

[0072] Figure 2 Evaluation of simulated single-cell samples labeled with IBT-16plex and TMTpro 16Plex in group B, containing 50-fold carriers. (A) Labeling efficiency. (B) Qualitative number of proteins in each labeled channel. (C) Box plot of quantitative accuracy for the two labeling reagents. (D) Comparison of method 1 before and after normalization. (E) Comparison of method 2 before and after normalization.

[0073] Figure 3Evaluation of simulated single-cell samples labeled with IBT-16plex and containing 50-fold carrier in group D. (A) Labeling efficiency. (B) Qualitative number of proteins per labeled channel. (C) Box plot of quantitative accuracy of IBT-16plex labeling reagent.

[0074] It can be seen that the analytical performance of the combination of IBT-16plex and TMTpro 16Plex labeling is better than that of single labeling. The labeling efficiency is slightly higher than that of single labeling. The number of protein identifications obtained by the two labeling methods is about the same. The number of protein identifications is slightly higher when labeled with single labeling reagent, but the throughput can be increased by about 100% when the labels are combined, which can greatly improve the mass spectrometry detection throughput.

[0075] Example 2: Single-cell proteomics application experiment

[0076] To apply the label-coupled quantitative method of this invention to the determination of actual single cells, we selected 293T cells. For specific experimental methods, please refer to Example 1.

[0077] The number of proteins identified per channel in a single cell, as shown below Figure 4 As shown. Figure 4 Twenty 293T single cells were labeled using a combination of IBT-16plex and TMTpro 16Plex labeling reagents, with single-needle loading. The graph shows the protein identification count for each labeled channel. The results are presented in three replicates.

[0078] For comparison, we also used single-labeling reagents to label single cells, such as using each channel of the TMTpro 16Plex-B group to label 293T single cells and using each channel of the IBT-16plex-B group to label HeLa single cells. The results are as follows. Figure 5 As shown, (A) represents the qualitative count of protein per labeled channel in TMT-labeled 293T single cells. (B) represents the qualitative count of protein per labeled channel in IBT-labeled HeLa single cells.

[0079] The comparison shows that when applied to actual cell testing, the number of protein identifications obtained by the two labeling methods is about the same. 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 100% when the labels are used in combination.

[0080] In the above embodiments, the coupled mass spectrometry analysis was based on a timsTOF SCP mass spectrometer. Due to the limitation of secondary spectral resolution, only half the throughput could be used per sample loading. If the TMTpro 16Plex and IBT16-Plex labeling coupling method of the present invention is analyzed using higher resolution mass spectrometers such as Orbitrap, Astral, timsTOF HT, and timsTOF Ultra, the detection throughput of single-cell samples can be increased by up to 30 times.

[0081] In summary, this demonstrates the feasibility of the IBT-16plex and TMTpro 16 Plex combination labeling scheme in this application. Applying it to the analysis and identification of single-cell proteomics can greatly improve efficiency, reduce costs, and also has high sensitivity.

[0082] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A high-throughput single-cell proteomics analysis method based on label-coupled techniques, wherein the method is based on tandem mass spectrometry labeling technology, characterized in that, The analytical method includes the following steps: (1) Extract single-cell proteins to obtain single-cell polypeptide samples; (2) Prepare several reaction containers, add single-cell polypeptide samples to each reaction container, add single labeling reagents with different tags to each single reaction container, mix with the polypeptide samples, react, terminate the reaction, centrifuge the samples in each single reaction container and mix them into a small tube to obtain mass spectrometry sample. The labeling reagents are TMTpro 16plex and IBT-16plex, wherein the label in TMTpro 16plex is set to the TMT group and the label in IBT-16plex is set to the IBT group. (3) Sample loading and mass spectrometry analysis were performed to obtain qualitative and quantitative protein data for each labeled channel; (4) The data of each marked channel obtained are processed using a normalization method; The normalization method is as follows: Normalization of conversion coefficients for each protein is achieved through the following steps: Based on the multi-sample combination, the quantitative search results of the IBT group and the TMT group are merged to obtain a commonly identified protein expression matrix. Quantitative data for each label channel of different groups are obtained from the merged protein matrix. Missing values ​​are filled using the minimum value. The mean of the quantitative results for different label channels of different proteins in the quantitative matrix is ​​obtained, forming a unique conversion coefficient between the TMT-IBT groups for different proteins. The protein conversion coefficients are then used to convert the quantitative results for each label channel of other samples. Alternatively, normalization can be performed using an additional reference channel. The steps are as follows: take an additional channel from each group of IBT and TMT as a reference channel, add the same amount of standard to the reference channel, and use the reference channel to correct other channels and / or multiple batches of samples to obtain the relative quantitative results.

2. The method according to claim 1, characterized in that, In step (2), the protein concentration in each individual reaction vessel is 0-5 ng / µL.

3. The method according to claim 1, characterized in that, In step (2), the concentration of the labeling reagent of a single label in each single reaction vessel is 0.5-5 µg / µL.

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

5. The method according to claim 1, characterized in that, The mass spectrometry used is high-resolution mass spectrometry.

6. The application of the label-based high-throughput single-cell proteomics analysis method according to any one of claims 1 to 5 in single-cell proteomics analysis and identification.

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