A proteomics standard and preparation method thereof

Abstract

The present invention discloses a proteomics standard product and a preparation method thereof, and specifically discloses a corresponding kit and a method for screening a proteomics standard product cell line. The standard product provided by the present invention is stable between generations, and after multiple passages, the quantitative analysis of the proteome is robust. The application of the standard product of the present invention can effectively improve the accuracy of proteomics quantitative analysis.

Description

Technical Field

[0001] The invention belongs to the field of proteomics, and in particular relates to a proteomics standard product and a preparation method thereof. Background Art

[0002] In recent years, with the rapid development of bio-omics research including genomics, transcriptomics, translationomics and proteomics, and driven by the cross-application of bioinformatics and big data science, a new medical concept and medical model, namely precision medicine, has been born.

[0003] Bio-omics research is one of the most important means to reveal disease mechanisms and precision medicine. The quality of omics data is directly related to the accuracy of disease mechanisms and the effectiveness of precision medicine. However, the current omics data are not uniform in the selection of standards by laboratories, and the sample processing methods are diverse, the measurement platforms are diverse, and the analytical techniques are imprecise, resulting in poor reproducibility and experimentality of cross-platform and cross-laboratory data. The Universal Human Reference RNA (UHRR) standard is a mixed RNA of 10 cancer cell lines including HeLa, and its instability is well known. In fact, in the comparison between UHRR batches, the Pearson correlation only reached R2 = 0.9478, indicating that this instability exists even in short production cycles. This variation is expected to increase exponentially in long production cycles and is insufficient to evaluate the reproducibility of next-generation sequencing technologies that are evolving with increasing depth and resolution. Hela cells, which have been used as standards in the past, have been widely debated for their strong intra-species and inter-species contamination. Studies have shown that Hela cell lines used in different laboratories were centrally cultured and continuously subcultured for 50 generations. Using the latest quantitative proteomics and other omics technologies, combined with molecular cytology experiments, it was found that Hela cells with the same name had significant biological variations at the phenotypic and molecular levels. Therefore, the selection of standards with robust consistency at multiple omics levels is the key to ensuring the stable output of group data. Summary of the invention

[0004] The present invention provides a proteomics standard product and a preparation method thereof by way of examples to solve the problem of instability between generations of existing standard products, and provides a stable standard product for the development of proteomics.

[0005] In order to achieve the above-mentioned purpose, the present invention is implemented according to the following technical scheme:

[0006] A proteomics standard product, which is a whole cell protein extracted from an MHCC97H cell line or a polypeptide obtained by hydrolyzing the whole cell protein.

[0007] Preferably, the proteome standard is prepared by the following method: step 1, culturing MHCC97H cells; step 2, extracting total protein from MHCC97H cells.

[0008] Preferably, the method further comprises step three; specifically, hydrolyzing the total protein obtained in step two to obtain polypeptides.

[0009] The present invention also provides a kit, which contains the proteomics standard product.

[0010] The present invention provides a method for preparing a proteomics standard; the method comprises the following steps:

[0011] Step 1, culturing MHCC97H cells;

[0012] Step 2: Extract and obtain total protein from MHCC97H cells.

[0013] Preferably, step 2 is specifically as follows:

[0014] 1. Dissolve the SDS lysis buffer and mix well; take an appropriate amount of lysis buffer and add PMSF before use to make the final concentration of PMSF 1mM;

[0015] 2. Remove the cell culture medium and wash once with PBS;

[0016] 3. Add SDS lysis buffer at a ratio of 150-250 μl of SDS lysis buffer per million cells, depending on the number of cells; pipette several times to ensure that the lysis buffer and cells are in full contact;

[0017] 4. Place the sample on ice for 30 minutes and vortex for 15 seconds every 10 minutes;

[0018] 5. Centrifuge at 17000×g, 4°C for 30 minutes, aspirate the supernatant as the proteomics standard and store in a -80° refrigerator.

[0019] Preferably, the method further comprises step three; the specific operation of step three is: performing filter-assisted sample preparation method enzymatic hydrolysis on the total protein obtained in step two to obtain polypeptides; specifically:

[0020] 1. Add protein denaturant urea to 200 μg of total protein sample in each tube, and make the final concentration of urea greater than 6M. If there is visible precipitation in the liquid, use an ultrasonic device to treat it for 20 seconds, pausing every 5 seconds;

[0021] 2. Add 50 mM DTT and place in a 37°C water bath for 1 hour;

[0022] 3. Add 100 mM IAA and let stand at room temperature for 30 min;

[0023] 4. Transfer the reduced and alkylated protein samples to a 30 kDa ultrafiltration tube and centrifuge at 12,000 × g for 20 min at 4 °C;

[0024] 5. Add 100 μL 8 M urea, centrifuge at 12,000 × g for 20 min at 4 °C, and repeat this step once;

[0025] 6. Add 100 μL 50 mM TEAB, centrifuge at 12,000 × g for 20 min at 4 °C, and repeat this step four times;

[0026] 7. Add 50 μL of 50 mM TEAB and then trypsin, where the ratio of protein to trypsin is 30:1;

[0027] 8. Place the ultrafiltration tube in a clean casing, seal the tube opening with sealing film, and incubate in a 37°C incubator for 12 hours;

[0028] 9. Centrifuge at 12000×g for 20 min at 4°C to collect peptide fragments to obtain proteomics standards in polypeptide form.

[0029] The present invention also provides a method for screening a proteomics standard cell line, comprising the following steps:

[0030] Step 1, culturing the target cells to be screened; specifically: using DMEM complete medium containing 10% fetal bovine serum, 1% double antibody, and 0.5% ciprofloxacin to culture the cells, the cells are digested with trypsin and passaged when the cells grow to 90% density, and 8 to 10 generations of cells are collected continuously, and 8×106 cells are collected each time for standby;

[0031] Step 2: extracting and obtaining total protein from the obtained cells of each generation; the specific steps are as follows:

[0032] 1. Dissolve the SDS lysis buffer and mix well; take an appropriate amount of lysis buffer and add PMSF before use to make the final concentration of PMSF 1mM;

[0033] 2. Remove the cell culture medium and wash once with PBS;

[0034] 3. Add SDS lysis buffer at a ratio of 150-250 μl of SDS lysis buffer per million cells, depending on the number of cells; pipette several times to ensure that the lysis buffer and cells are in full contact;

[0035] 4. Place the sample on ice for 30 minutes and vortex for 15 seconds every 10 minutes;

[0036] 5. Centrifuge at 17000×g, 4°C for 30 minutes, aspirate the supernatant as the total protein sample, and store in a -80° refrigerator;

[0037] Step 3: The total protein obtained in step 2 is hydrolyzed by filter-assisted sample preparation method to obtain polypeptides; the specific operation is as follows:

[0038] 1. Add protein denaturant urea to 200 μg of total protein sample in each tube, and make the final concentration of urea greater than 6M. If there is visible precipitation in the liquid, use an ultrasonic device to treat it for 20 seconds, pausing every 5 seconds;

[0039] 2. Add 50 mM DTT and place in a 37°C water bath for 1 hour;

[0040] 3. Add 100 mM IAA and let stand at room temperature for 30 min;

[0041] 4. Transfer the reduced and alkylated protein samples to a 30 kDa ultrafiltration tube and centrifuge at 12,000 × g for 20 min at 4 °C;

[0042] 5. Add 100 μL 8 M urea, centrifuge at 12,000 × g for 20 min at 4 °C, and repeat this step once;

[0043] 6. Add 100 μL 50 mM TEAB, centrifuge at 12,000 × g for 20 min at 4 °C, and repeat this step four times;

[0044] 7. Add 50 μL of 50 mM TEAB and then trypsin, where the ratio of protein to trypsin is 30:1;

[0045] 8. Place the ultrafiltration tube in a clean casing, seal the tube opening with sealing film, and incubate in a 37°C incubator for 12 hours;

[0046] 9. Centrifuge at 12000×g for 20 min at 4°C to collect peptide fragments to obtain proteomics standards in peptide form;

[0047] Step 4: Mass spectrometry identification: The peptide samples of each generation obtained above are subjected to mass spectrometry quantitative identification; the expression levels of the proteomes of each generation are obtained by searching the library;

[0048] Step 5. Proteomic quantitative stability assessment: perform correlation analysis on the proteomic expression levels of different generations of the same cell line to obtain the Pearson correlation coefficient; compare the correlation coefficients between different cell lines, and select the cell line with the highest correlation coefficient as the cell line for preparing proteomic standards.

[0049] In the screening method described in the present invention, the correlation coefficients of multiple cell lines can be directly compared horizontally as described above, or the correlation of transcriptome data can be used to perform a primary screening to obtain a relatively stable cell line at the transcriptomic level; then the proteomics correlation coefficient analysis between the generations of the selected target cell line is performed. If the correlation coefficient is high, such as greater than 0.9, the cell line is considered stable and can be selected as a standard cell line. The reproducibility of proteomics data was relatively poor in the past. The correlation R2 of the proteomics of the present invention can be achieved above 0.9, which has a qualitative leap relative to past data; the protein quantitative correlation of the MHCC97H cell line of the present invention can even reach 0.98 and 0.99. Past proteomic quantitative data is considered to be at a very high level as long as the correlation reaches above 0.8; so the correlation of the present invention has far surpassed almost all data sets in the field; there is no need to compare horizontal cell lines.

[0050] The beneficial effect of the present invention is that: the present invention continuously collects 5 cell lines of human liver cancer cell lines MHCC97H, HCCLM3, HCCLM6, human lung cancer cell line A549, and human cervical cancer cell line Hela, and performs proteomic detection to obtain the cell line with the highest proteomic stability during cell passage, thereby selecting the cell line with the highest stability as the candidate reference material collection object, screening out the reference cell line with high robustness in the proteomic level as a standard that can be widely promoted, and using this to develop a translation assembly library and analysis process for slightly degraded samples, thereby increasing the robustness of such samples. The present invention shows a human cell line with a stable proteomic across multiple passages and its corresponding standard, which is suitable as a material for long-term production of reference proteomic standard substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to clearly demonstrate the specific implementation methods of the present invention and certain detection technologies used in the experiments, the implementation methods and the technologies used will be described below, mainly in the form of drawings.

[0052] Figure 1 .Quantitative stability assessment of transcriptomes of five cell lines at different generations. Figures A to E are quantitative correlation analysis of transcriptomes of five cell lines at different generations, namely MHCC97H, HCCLM6, HCCLM3, Hela and A549, with the horizontal and vertical axes representing different cell generations; Figure F shows the technical repetition of three transcriptome sequencing of MHCC97H cells at the 3rd, 5th, 7th and 9th generations, respectively.

[0053] Figure 2.Stability assessment of multi-center, multi-temporal sequencing of the MHCC97H transcriptome. Two batches of RNA from different generations of MHCC97H cell lines were obtained at different times (May and December 2021), and transcriptome libraries were constructed and sequenced, and quantitative correlation analysis was performed. Figure A shows the quantitative correlation analysis of transcriptome sequencing in December 2021; Figure B shows the quantitative correlation comparative analysis of the corresponding samples in December and May. After RNA from different generations of MHCC97H cell lines was extracted, it was divided into two and handed over to two experimenters with 2 years of high-throughput sequencing experience for transcriptome library construction and sequencing. Figure C shows the quantitative correlation analysis results of transcriptome sequencing of different generations of MHCC97H obtained under the operation of experimenter 2; Figure D shows the quantitative correlation comparative analysis results of transcriptome sequencing of MHCC97H by experimenter 1 and experimenter 2. After RNA extraction from MHCC97H cell lines of different generations, the cells were divided into two groups. The RNA samples of MHCC97H were sent to Chengqi Biotechnology and Saizhe Gene respectively. Figures E and F are the results of quantitative correlation analysis of transcriptome sequencing by two commercial companies, and the horizontal and vertical coordinates are cell generations. Different sequencing data alignment algorithms were used to analyze the transcriptome sequencing data of different generations of MHCC97H in the same batch to obtain quantitative correlation analysis data. Figure H uses the Bowtie2 algorithm, and Figure I uses the Hista algorithm. Two different ribosomal RNA removal processes were used for transcriptome sequencing. Figure J is the correlation analysis result of MHCC97H transcriptome data measured after removing ribosomal RNA using the Ribominus method. Figure K is the comparative analysis result of quantitative correlation of MHCC97H transcription sequencing by PolyA+mRNA enrichment method and Ribominus method.

[0054] Figure 3 .Evaluation of the library construction method for naturally degraded RNA samples. Figure A: RNA degradation of MHCC97H cells of different generations caused by simulated environmental exposure, and agarose gel electrophoresis of degraded RNA; Figures B to D use different algorithms (FANSe, bowtie2 and Hista) to perform quantitative correlation comparison analysis on the sequencing data of RNA samples of different generations of MHCC97H degraded and non-degraded RNA samples constructed by polyA+mRNA enrichment method, normal represents non-degraded RNA samples, and degradation represents degraded samples.

[0055] Figure 4 The quantitative stability evaluation of MHCC97H proteome. Figure A shows the quantitative correlation analysis results of MHCC97H translation group sequencing at different generations; Figure B / C shows the quantitative correlation analysis results of proteomics collected by MHCC97H cells at different generations in DDA and DIA modes; Figure D / E shows the quantitative correlation analysis results of proteomics of three independent experiments in DDA and DIA acquisition modes of MHCC97H cells at the third generation.

[0056] Figure 5 This is a quantitative correlation analysis of the MHCC97H proteome in different laboratories. Figure A shows the mass spectrometer, chromatographic model, HPLC column and analytical column specifications used by the four experiments; Figure B shows the isoelectric point distribution diagram of proteins identified by different laboratories and the P value results obtained by pairwise KS analysis; Figures C / D show the correlation analysis results of proteins identified by different laboratories in the same sample under DDA and DIA modes.

[0057] Specific implementation methods

[0058] The specific implementation methods of the present invention are explained with the aid of examples. Except that the technology used for detection does not impose any form of limitation on the present invention, some schemes in the described embodiments belong to part of the embodiments of the invention. The embodiments obtained by ordinary technical operators in this field without creative results all belong to the protection scope of the present invention.

[0059] Example 1

[0060] Main reagents and formula

[0061] Cell lines

[0062] Human hepatoma cells MHCC97H, HCCLM6 and HCCLM3 were purchased from the Institute of Biomedical Sciences, Fudan University; MHCC97H cells are common and easily available, and the origin of this cell line is disclosed in several authorized patents (CN107523544B, CN102813643B). Human cervical cancer Hela cells and human non-small cell lung cancer cells A549 were purchased from the American Type Culture Collection.

[0063] Cell culture reagents

[0064] Dulbecco's modified eagle medium (DMEM), Life Technology, USA;

[0065] Fetal bovine serum (FBS), Life Technology, USA;

[0066] Penicillin-streptomycin, PS, Guangzhou Jiebei Biotechnology Co., Ltd.

[0067] Pancreatin, Amresco, USA;

[0068] Ciprofloxacin (CIP), Guangzhou Nanxin Pharmaceutical Co., Ltd.;

[0069] Dimethylsulfoxide (DMSO), Sigma, USA;

[0070] T25, T75, and T175 cell culture flasks, Becton Dickison, USA;

[0071] L-Glutamine, Sigma, USA;

[0072] Main instruments

[0073] Analytical balance, Syngene, UK;

[0074] MilliPore pure water system, MilliPore, USA;

[0075] Clean bench, Jiangsu Taisujing Antai Company, China;

[0076] Small high-speed refrigerated centrifuge, Eppendorf, Germany;

[0077] Ice maker, Sanyo Co., Japan;

[0078] Optima L-XP ultracentrifuge, Beckman, USA;

[0079] BGIseq500 / 2000 sequencer, BGI Intelligent Manufacturing Co., Ltd., China;

[0080] Protein Enzymatic Reagents

[0081] Sodium-dodecyl sulphate (SDS) and urea, Guangzhou Jiebeis Biotechnology Co., Ltd.;

[0082] KH2PO4, NaH2PO4·H2O and KCl, Tianjin Damao Chemical Reagent Factory;

[0083] Dithiothreitol (DTT) and iodoacetamide (IAA) were from Sigma, USA;

[0084] Triethylammonium bicarbonate (TEAB) buffer and formic acid, Fluka, USA;

[0085] Acetonitrile and Bicinchoninic acid (BCA) protein concentration determination kit, ThermoFisher China Co., Ltd.;

[0086] 30 kDa ultrafiltration tube, Millipore, Germany;

[0087] Trypsin for protein enzymatic hydrolysis, Promega, USA;

[0088] Protein extraction and enzymatic hydrolysis reagent formula

[0089] 1. SDS protein lysis buffer: purchased from Shanghai Bio-Tech Biotechnology Co., Ltd., the main components are: 50mM Tris (pH 8.1), 1% SDS and Na4P2O7·10H2O, β-glycerophosphate, Na3VO4, NaF, EDTA and leupeptin and other protease inhibitors;

[0090] 2. Protein denaturant: 4% SDS, 8M urea;

[0091] 3. Protein reducing agent: 50mM DTT dissolved in 8M urea;

[0092] 4. Alkylating agent: 100 mM IAA dissolved in 8 M urea;

[0093] 5. Dissolution solution: 100 mM TEAB buffer;

[0094] 6. Trypsin digestion solution: Use 50mM TEAB to dissolve trypsin to a final concentration of 1μg / μL;

[0095] 7. HPLC eluent: Prepare 97.5% ddH2O, 2.5% acetonitrile, and 0.1% formic acid as solution A; 2.5%

[0096] ddH2O, 97.5% acetonitrile, and 0.1% formic acid were used to prepare solution B;

[0097] Cell culture

[0098] The cells were cultured in DMEM complete medium containing 10% fetal bovine serum, 1% double antibody, and 0.5% ciprofloxacin. The cells were digested with trypsin and passaged when they grew to 90% density. Cells were collected for 8 generations in succession, and 8×10 6 The number of cells was used for DDA and DIA proteomic experiments. The cells involved in this patent have been tested for mycoplasma and cell STR, and no mycoplasma contamination or cell cross-contamination was found.

[0099] RNA extraction and transcriptome sequencing library construction

[0100] The library construction and sequencing process were carried out according to the human poly-AmRNA standard transcriptome. For specific experimental steps, please refer to the applicant's published literature [1,2].

[0101] The total RNA extraction process is as follows: when the cells have grown to fill the 75cm2 culture flask, discard the culture supernatant, place on ice, and wash twice with pre-cooled PBS. Discard PBS and add 5mL pre-cooled Trizol (total RNA extraction reagent) for extraction. For specific steps, refer to the Trizol instruction manual.

[0102] The process of enriching total mRNA uses the VAHTS mRNA CaputreBeads to enrich poly-AmRNA. For specific steps, see the instructions:

[0103] Transcriptome library construction from degraded RNA samples

[0104] Use the Ribo-off rRNA depletion kit (Human / Mouse / Rat) to build a library for degraded RNA samples. For specific steps, please refer to the instruction manual of the product.

[0105] The library was constructed using the MGI transcriptome library construction kit. PE150 sequencing was performed using the BGIseq2000 high-throughput sequencing platform. The sequencing data was aligned to the human transcriptome reference sequence using FANSe3 with the parameters -L80-E5-I0-S14-B1-U0.

[0106] Analysis parameters of different mapping algorithms in this patent

[0107]

[0108] Protein extraction

[0109] Protein extraction was performed according to the instructions for protein extraction of cell samples with SDS protein lysis buffer from Bio-Tech. The specific operation method is as follows:

[0110] 1. Dissolve the SDS lysis buffer and mix well. Take an appropriate amount of lysis buffer and add PMSF within a few minutes before use to make the final concentration of PMSF 1mM;

[0111] 2. Remove the cell culture medium and wash once with PBS;

[0112] 3. Add lysis buffer at a ratio of 150-250 μl per 1 million cells, depending on the number of cells. Use a gun to blow several times to ensure that the lysis buffer and cells are in full contact;

[0113] 4. Lyse on ice for 30 minutes. Vortex for 15 seconds every 10 minutes.

[0114] 5. Centrifuge at 17000×g, 4°C for 30 minutes, and take the supernatant as the protein standard. Store in a -80° refrigerator.

[0115] If necessary, the protein concentration can be measured using a BCA protein concentration quantification kit.

[0116] Filter-aided sample preparation (FASP) enzymatic hydrolysis 1. Add an appropriate volume of protein denaturant to 200 μg of protein sample per tube and make the final urea concentration greater than

[0117] At 6M, if there is visible precipitation in the liquid, use 35% power ultrasound for 20s, pausing every 5s;

[0118] 2. Add 50 mM DTT and place in a 37°C water bath for 1 hour;

[0119] 3. Add 100 mM IAA and let stand at room temperature for 30 min;

[0120] 4. Transfer the reduced and alkylated protein samples to a 30 kDa ultrafiltration tube and centrifuge at 12,000 × g for 20 min at 4 °C;

[0121] 5. Add 100 μL 8 M urea, centrifuge at 12,000 × g for 20 min at 4 °C, and repeat this step once;

[0122] 6. Add 100 μL 50 mM TEAB, centrifuge at 4°C, 12,000 × g for 20 min, and repeat this step four times; 7. Add 50 μL 50 mM TEAB and then trypsin, where the ratio of protein to trypsin is 1:30;

[0123] 8. Place the ultrafiltration tube in a clean casing, seal the tube opening with sealing film, and incubate in a 37°C incubator for 12 hours;

[0124] 9. Centrifuge at 12000×g for 20 min at 4°C to collect peptide fragments.

[0125] 10. The lyophilized peptide samples were stored in a -80℃ refrigerator.

[0126] Reversed-phase liquid chromatography combined massspectrum (RPLC-MS)

[0127] 1. Dissolve the peptide in 10 μL of liquid and use a reverse phase column Durashell C 18 (L)(2.1mm×

[0128] 50mm)(particle size: 3μm; pore size: ) was used for pre-separation of components by Eksigent chromatograph. The chromatographic separation time was 45 min, and six components were obtained from each sample. The specific gradient of mobile phase separation was

[0129] See the table below:

[0130]

[0131] 2. Freeze-dry the peptide components obtained in the pre-separation, dissolve the peptide powder in 25 μL of liquid, and heat at 4°C, 12000

[0132] × g centrifugation for 20 min, and 10 μL of the sample was taken for MS identification;

[0133] 3. The specific parameters of the Orbitrap Fusion mass spectrometer were set as follows: the cycle time was set to 3.5 s, the resolution of MS1 was 120 K, and the scan range of MS1 was 350-1550; the resolution of MS2 was set to 15 K, the ion fragmentation mode was HCD, the dynamic exclusion was 45 s, n = 1, the AGC threshold was 5e4, and the maximum injection time was 90 ms;

[0134] Get the raw file, which is the original proteomic data.

[0135] Database Search

[0136] The protein identification retrieval software Maxquant1.6.2 was used for qualitative and quantitative analysis of protein database searches. The database used had the following search parameters: the tolerance value for MS1 was 10 ppm, the tolerance value for secondary fragments was 0.02 Da, the maximum number of missed cuts allowed was 2, Carbamidomethyl (cysteine) was fixed modification, methionine oxidation (methionine), and acetylation (protein N-terminus) were variable modifications, protein identification controlled peptide level FDR < 0.01, protein level FDR < 0.01, and specific peptides were at least greater than 1.

[0137] References: If there are any incomplete details of the methods used in the present invention, please refer to the following two references.

[0138] 1.Wang,T.,etal.,TranslatingmRNAs stronglycorrelatetoproteins inamultivariatemannerandtheirtranslationratios arephenotypespecific.NucleicAcidsRes,2013.41(9):p.4743-54.

[0139] 2.Li,D.,et al.,Optimal Settings ofMass Spectrometry Open SearchStrategy for Higher Confidence.JProteome Res, 2018.17(11):p.3719-3729.

[0140] Example 2 Results and Discussion

[0141] HCC97H can be used as a stable source cell line for transcriptome standards

[0142] Discovery and screening of potential stable reference cell lines

[0143] The present invention tested five commonly used cell lines, MHCC97H, HCCLM6, HCCLM3, HeLa and A549. For each cell line, the present invention cultured for 8 to 11 generations and sampled from each generation. Total RNA was extracted from each sample, and the RNA quality was checked by electrophoresis to verify that they were not degraded. PolyA+mRNA was then sequenced and quantified using the RPKM method. Cross-correlation analysis showed that the transcriptome of MHCC97H was the most stable, with Pearson R 2 =0.966~0.995( Figure 1 A). The other two HCC cell lines had lower consistency (R 2 can be as low as 0.947 and 0.921 respectively, Figure 1 B-C). The consistency between generations of HeLa and A549 cells was even lower (R 2 As low as 0.899 and 0.846 respectively, Figure 1 D~E). To be precise, this deviation includes biological deviation (inconsistency of transcriptome between generations) and experimental error (errors caused by library construction, sequencer and data processing algorithm). The present invention also verifies the experimental error by sequencing the same MHCC97H RNA sample of different generations three times independently, and obtains the average R 2 =0.98 Pearson correlation ( Figure 1 F) This shows that Figure 1 The deviation shown in A can almost be explained by experimental error, i.e., the biological deviation of MHCC97H is almost negligible. In contrast, the R 2 The lower limit decreases with the passage of generations ( Figure 1 B-E), indicating that their biological differences increased significantly with the passage of generations and therefore they are not ideal candidates for standards.

[0144] MHCC97H transcriptome stability assessment

[0145] A good reference standard should be easy to produce and complemented by a robust standard processing protocol that, ideally, produces consistently identical results. The main steps that introduce bias are cell culture, mRNA enrichment, library construction / sequencing, and data processing algorithms. First, two batches of the MHCC97H cell line were subcultured in May and December 2021, respectively. The generations of gene expression were similar, indicating that the cell cultures were consistent. Secondly, the robustness of the process was tested by different experimenters and different laboratories. The same series of total MHCC97H RNA was used as the starting material, and another experimenter was asked to independently construct the library using another batch of library construction kits. Results ( Figure 2 C / D) and the former experimenter ( Figure 1 A) is almost the same. In addition, the present invention also sent 4 samples to two commercial sequencing service providers more than 1,000 kilometers away. Chi-Biotech Co. Ltd. is equipped with MGISEQ-2000 sequencer, and Sagene Co. Ltd. is equipped with Illumina NovaSeq-6000 sequencer. They used Illumina kits to construct libraries and operated the sequencers according to the manufacturer's instructions. R 2 The coefficients were 0.959-0.981 and 0.941-0.981 ( Figure 2 E~F). The quantitative correlation analysis was performed on the sequencing data of the two commercial companies and the data generated by Jinan University. The quantitative correlation of the three laboratories for the same sample was extremely high ( Figure 2 G).

[0146] Next, the present invention studied the impact of mapping and quantification algorithms on robustness. The standard SOP of the present invention used the FANSe3 algorithm and the RPKM method to quantify the gene expression of the MHCC97H subculture dataset, achieving R 2 =0.966~0.995( Figure 1 A). We also tested two widely used RNA-seq algorithms, Bowtie2 and HISAT, which showed lower R 2 =0.963~0.991( Figure 2 H,I), indicating that these algorithms are not as robust as FANSe.

[0147] Finally, the present invention tested different mRNA enrichment strategies. The standard protocol of the present invention uses oligo-dT to enrich polyA+mRNA (mature mRNA), which is used in most studies. Another strategy is the rRNA depletion strategy (also known as ribo-reduction), which removes rRNA by probe hybridization or RNaseH method. When the rRNA depletion strategy is used, Pearson R 2 Coefficient = 0.925 ~ 0.971 ( Figure 2 K, J), which is much lower than the polyA+ method.

[0148] Degradation of MHCC97H RNA can achieve higher transcriptome quantitative stability

[0149] A good RNA reference standard should have high robustness in actual use, and always obtain the same results even if inevitable degradation occurs. The ribonucleases commonly present in the experimental environment can quickly degrade RNA. In addition, since RNA standard samples may degrade to varying degrees during long-distance logistics transportation or storage, the standard may be inaccurate. In order to confirm whether the RNA of MHCC97H cells still has good stability after degradation, the present invention also tests such degraded RNA standard samples. First, the present invention creates a scenario that simulates degradation caused by environmental exposure: the RNA sample of MHCC97H is exposed to the air for a long time, so the RNA enzymes in the environment may enter the test tube and degrade the RNA. This is the most common scenario in actual experiments. Electrophoresis shows that the total RNA of MHCC97H has different degrees of degradation ( Figure 3 A). The present invention uses the oligo-dT method to enrich mRNA. Surprisingly, the SOP developed for non-degraded samples can be directly used for degraded samples without any experimental and analytical changes, and can produce highly comparable gene expression values ​​(Pearson R 2 >0.91)( Figure 3 B, C, D).

[0150] MHCC97H can be used as a stable source cell line for protein standards

[0151] Quantitative stability assessment of the MHCC97H proteome

[0152] The present invention has found that MHCC97H cells have the highest stability among 5 different cell lines through transcriptome technology. In order to further illustrate the stability of MHCC97H at the proteomic level, the present invention tested the translation group samples of MHCC97H of consecutive generations and found that the quantitative correlation of MHCC97H translation group still reached 0.948-0.991 ( Figure 4A), indicating that the stability of the MHCC97H translation group level is extremely high. Since the correlation between the translation group and the proteome is extremely high, it indirectly indicates that the stability of the MHCC97H proteome may be relatively high. The present invention further performs mass spectrometry identification on the whole protein of the 2nd to 9th generation cells of MHCC97H, and uses an Orbitrap Fusion Lumos mass spectrometer to identify the peptide fragments, forming 9 DDA and 9 DIA data sets. The target-decoy library search strategy is used to reduce the false positive rate of identification, and finally a qualitative and quantitative list of proteins between cells of different generations is obtained. The quantitative results of the 2nd to 9th generation cell proteins of MHCC97H were subjected to correlation analysis, and the quantitative correlation between the proteins of each generation under the DDA mode reached R 2 =0.934-0.976( Figure 4 B), the quantitative correlation between the generations of proteins under DIA mode reached R = 0.942-0.986 ( Figure 4 C); The present invention used the third generation MHCC97H cells to perform three independent enzymatic hydrolysis experiments, and the quantitative correlation under the DDA mode was 0.945-0.949 ( Figure 4 D), the quantitative correlation in DIA mode is 0.975-0.99 ( Figure 4 E). The quantitative correlation of nucleic acid and protein between each generation of the candidate standard MHCC97H reached an ultra-high level, showing high stability at both the nucleic acid and protein levels, further indicating that the MHCC97H cell line can be used as a protein standard sample for continuous and stable production.

[0153] Consistency evaluation of mass spectrometry in third-party laboratories for MHCC97H proteome samples

[0154] Good protein standards should achieve similar identification and quantification effects in different laboratories and have high stability. The present invention gives 1 mg of protein from the same batch of standard sample MHCC97H cell line to each laboratory of South China University of Technology (SCUT), Beijing Normal University (BNU), Dalian Institute of Chemical Physics, Chinese Academy of Sciences (DICP) and Jinan University (JNU). All laboratories adopt standardized enzymatic hydrolysis and mass spectrometry experimental processes. The quality control method is as follows: the ultrafiltration tube enzymatic hydrolysis standard operation is adopted for the experiment. After enzymatic hydrolysis, the peptide is desalted and the peptide concentration is tested. After enrichment, the peptide is ≥1μg / μL. The instruments of the three laboratories are all unified as Thermo Orbitrap Fusion Lumos, and one uses Q Exactive plus ( Figure 5A). The target-decoy library search strategy was used to reduce the false positive rate of identification, and finally a qualitative and quantitative list of proteins from different laboratories for the same sample was obtained. Pairwise KS statistical analysis of the isoelectric point distribution of proteins identified by different laboratories did not yield statistically significant differences (P value > 0.05) ( Figure 5 B), indicating that the isoelectric point distribution of the same sample protein in the four laboratories is relatively consistent. The DDA iBAQ quantitative consistency test of the protein quantitative results of the four laboratories is the same as the laboratory quantitative repeatability R 2 =0.925-0.949( Figure 5 C), correlation R between DIA quantitative data from different laboratories 2 = between 0.832 and 0.926 ( Figure 5 D) It should be noted that the correlation is lower than that of the other three experiments because the mass spectrometer model used by SCUT is different from that of the other three. Under mass spectrometry identification in different laboratories, the protein mass spectrometry quantification of the MHCC97H cell line still achieves ultra-high repeatability and verifiability.

[0155] in conclusion

[0156] The present invention collects 5 cell lines such as MHCC97H for multiple generations and performs transcriptome sequencing. It is found that among the 5 experimental cell lines, the quantitative correlation of MHCC97H cell transcriptome at the 5th to 14th generations is the highest. 2 The results showed that the transcriptome stability of MHCC97H cells was the highest during cell culture, and the stability of the translation group of the MHCC97H cell line was further evaluated. The results showed that the RNC-seq quantitative correlation R of the MHCC97H cell line reached above 0.9, and the protein mass spectrometry quantitative result R also reached above 0.92. In addition, repeatable and verifiable stable data were obtained in a third-party laboratory according to the standard process. This result shows that MHCC97H is very stable, can be used as a standard substance for long-term production, and can be widely promoted and used in various laboratories.

[0157] In addition, the results obtained from the research of the present invention are based on five cell lines, namely MHCC97H, HCCLM3, HCCLM6, A549, and Hela. It can only be said that the stability of the standard MHCC97H selected from these five experimental strains under a unified standard operating procedure is the strongest. The reason why the MHCC97H cell line is more stable than other experimental cell lines may be that during the process of multiple generations of continuous culture of cells, the MHCC97H cell line grows relatively slowly and has a lower basal metabolic rate than other experimental strains, which may be the reason for its higher stability.

[0158] The present invention evaluates the qualitative and quantitative accuracy of the MHCC97H cell line by performing a passage consistency test in 5 cancer cell lines. The results show that the MHCC97H cell line has super high stability and can be stably produced as a standard product for the output of omics data. The present invention also optimizes the unified sequencing process and mass spectrometry process and has super high robustness, which can be widely transplanted and promoted in various laboratories. In short, the research of the present invention aims to develop key technologies and application demonstrations for the quality control of each type of life omics data, establish standard methods and automated tools for the quality control of life omics data throughout the process, promote the application of life omics data in basic medicine and clinical medicine, and accelerate the effective conversion and practical application of life omics data in the field of precision medicine, so that patients with various diseases can benefit. At the same time, the international influence of my country's life omics technology will be further enhanced, making precision medicine an important part of the country's strategic emerging industries.

[0159] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Application of whole cell proteins extracted from MHCC97H cell lines or peptides hydrolyzed from whole cell proteins of MHCC97H cells in the preparation of proteomics standards.

2. The use according to claim 1, characterized in that: The proteomics standard is prepared by the following method: step 1, culturing MHCC97H cells; step 2, extracting total protein from MHCC97H cells.

3. The use according to claim 2, characterized in that: The method further comprises step three, specifically: hydrolyzing the total protein obtained in step two to obtain polypeptides.

4. The use according to claim 3, characterized in that: The specific operations of step 2 are:

1. Dissolve the SDS lysis buffer and mix well; take an appropriate amount of lysis buffer and add PMSF before use to make the final concentration of PMSF 1mM; 2. Remove the cell culture medium and wash once with PBS; 3. Add SDS lysis buffer at a ratio of 150-250 μl of SDS lysis buffer per million cells, depending on the number of cells; pipette several times to ensure that the lysis buffer and cells are in full contact; 4. Place the sample on ice for 30 minutes and vortex for 15 seconds every 10 minutes; 5. Centrifuge at 17000×g, 4℃ for 30 minutes, aspirate the supernatant as the proteomics standard; store in a -80℃ refrigerator.

5. The use according to claim 4, characterized in that: The specific operation of step three is: the total protein obtained in step two is subjected to filter-assisted sample preparation method enzymatic hydrolysis to obtain polypeptides; specifically:

1. Add protein denaturant urea to 200 μg of total protein sample in each tube, and make the final concentration of urea greater than 6M. If there is visible precipitation in the liquid, use an ultrasonic device to treat it for 20 seconds, pausing every 5 seconds; 2. Add 50 mM DTT and place in a 37°C water bath for 1 hour; 3. Add 100 mM IAA and let stand at room temperature for 30 min; 4. Transfer the reduced and alkylated protein sample to a 30 kDa ultrafiltration tube and centrifuge at 4°C, 12,000 × g for 20 min; 5. Add 100 μL 8 M urea and centrifuge at 4°C, 12,000 × g for 20 min. Repeat this step once; 6. Add 100 μL 50 mM TEAB, centrifuge at 12,000 × g for 20 min at 4 °C, and repeat this step four times; 7. Add 50 μL 50 mM TEAB and then trypsin, where the ratio of protein to trypsin is 30:1; 8. Place the ultrafiltration tube in a clean cannula, seal the tube with a sealing film, and incubate in a 37°C incubator for 12 hours; 9. Centrifuge at 12000×g for 20 min at 4°C to collect peptide fragments to obtain proteomics standards in polypeptide form.

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