Quantitative Proteomics Based On Mass Spectrometry And Its Application In Colorectal Cancer

We developed and improved the methylation isotope labeling methods in quantitative proteome, and applied it in colorectal cancer (CRC) early diagnosis, lymphatic metastasis and protein post-translational modification research. The aim of our study is to interpret molecules events in the early stage of CRC and metastasis in CRC that may bring about new biomarkers for early diagnosis.For the first time, we combined methylation isotope labeling with methylation isotope labeling assisted gel enhanced liquid chromatography mass spectrometry (GeLC-MS) methods in quantitative proteome analysis, established standard methylation quantification pipeline and developed in house methylation quantification software that achieved high reproductive and precise relative methylation quantification. Furthermore, by introducing GeLC-MS strategy, we can not only quantify the protein expression difference between two samples, but also qualify and quantify their difference in degradation level which is rather important in cancer research. In our research, altogether 501 differentially expressed proteins between early stage CRC and normal tissues were discovered (P<0.05). In the most regulated 80 proteins, we found degradation in 20 of them. By functional annotation analysis, two glycoproteins, Al AT and CTSD which are playing important role in ubiquitination proteasome pathway were chosen as candidate biomarkers. On serum level, we confirmed the key role AlAT and CTSD playing in early CRC by tissue microarray and genetic experiments. By combed applying the two biomarkers,96.77%CRC patients were specifically detected.Quantitative proteomics based on Mass Spectrometry was introduced to CRC lymphatic metastasis research for the first time, and revealed the different expression level of metastatic potential related proteins. By using bioinformatics tools, we interpreted the differential proteome data to get candidate biomarkers for future functional analysis and validated them by Q-PCR. Change in protein post-translational modification in cancer is also an important molecule event in promoting tumor growth and cancer metastasis. We identified and compared the differential expressed glycosylated and phosphorylated proteins in normal and early CRC tissue, and thus established theory foundation for future CRC biomarker in clinical application.Part 1. Quantification Proteomics Research Based on Methylation Isotopic LabelingIn the field of mass spectrometry based proteome quantification, isotopic labeling has an advantage over other quantification methods for its high efficiency, high accuracy and high sequence coverage, but expansive isotope labeling reagents and complex isotope labeling methods restrict their application in quantification proteomics. To eliminate such constraint, we introduced a relatively cheap esterification reaction to isotopic labeling and optimized its performance in proteome quantification research. We d0-or d3-methylated the tryptic peptides of standard proteins, mixed them by different ratios, separated by LC and identified by mass spectrometry in order to evaluate the application of methylation reaction in LC-LTQ-Orbitrap pipeline. The spectrum of standard peptides showed that the methylation reaction was fast and completed without any detectable byproducts. High coefficient of determination under ratio 10 indicates good fidelity between theoretical and observed heavy/light ratio. Taking spectrum complexity and labeling efficiency into account, peptides with more than 6 reaction sites or charge state≥4+(5%of total spectrums) were not used. Based on above, we summarized a set of standards for selecting the candidate peptide for quantification by our quantification software. Good linearity and reproducibility indicate well qualified methylation labeling for quantification study.Part 2. Clinical diagnosis biomarker for Early Stage of CRC by Proteomics MethodCRC is the second highest probability of cancer death in the world and the forth in China while the probability keeps increasing with the improvement of people's life. In human life, chance of suffering from CRC is about 6%, and if diagnosed at early stage, 90%of the patients can be cured by surgery. Unfortunately, most CRC are diagnosed at advanced stage. Common biomarkers, such as carcinoembryonic antigen (CEA) lack enough sensitivity and specificity. Exploring more reliable biomarkers and new diagnostic tools is the only way for the development of CRC treatment.Given the grim situation CRC early diagnosis, we established and standardized proteomics techniques can be applied to post-translational modification peptide analysis, but the content of the PTM proteins are relatively low which requires the enrichment step before MS detection. The enzymatic peptides were methylation isotopic labeled before using immobilized metal ion affinity chromatography (IMAC) and titanium dioxide to enrich the phosphorylated peptides. Methylation labeling reaction enclosed the acide sites of the peptides that can increase the efficiency of the enrichment, and the specificity of the phosphorylation enrichment went up to 83.33%. In addition, we also found that, IMAC had better enrichment specificity than titanium dioxide while the latter could enrich more phosphorylated peptides. Further, the new core-shell nano-material was introduced to the glycolpeptide enrichment which identified 194 glycopeptide of 155 glycoproteins. The PTM change of CRC cancer can evaluate the development and treatment of the cancer.Part 5. Profiling Protein Expression in Chinese Healthy Liver by Quantitative Immunology ResearchHuman Liver Proteome Project (HLPP) is initiated by the Human Proteome Organization (HUPO) which is the first human organ-specific proteome research project aiming at establishing expression profile of Chinese healthy liver in order to obtain human liver protein expression, classification, network-related functions. The most comprehensive obstacle in science community is the large-scale high-throughput quantitative analysis of proteins, and HLPP used optimized peptide mass spectra counting method for MS semi-quantitation, and by three isolation and identification techniques,6788 non-redundant proteins were identified 7 orders of abundance magnitude on 95%confidence level. To demonstrate the accuracy of our quantitative mass spectrometry method, we did protein immunoassay on selected proteins from every abundance magnitude. For the choice of the antigen-antibodies, we first compared a list of commercial antibodies with our selected proteins and deleted antigens with extreme molecular weight (> 150Da and<20Da), and bought paried antigen-antibody. In the enzyme-linked immunoassay (ELISA) and immunoblotting (WB) test, the antigen (standard protein) and liver protein extracts each were taken a corresponding concentration gradient, and repeated until the intensity of liver protein fell between the standard protein concentration gradient, while the absolute amount of the protein was obtained by making standard curve. Meanwhile, for a small number of concentration magnitude which lacked immunology data, we used additional standard chromatographic analysis which the content of the protein in the liver samples was assessed by the intensity of the UV absorption. Despite of two set of large-deviation data, absolute amount of proteins obtained by immunological methods, in general, validated peptide mass spectra of semi-quantitative count data, and provided important reference to the protein database of Chinese healthy liver.Part 6. Application of Increasing the Number of Charge State to Improve the Efficiency of Electron Transfer Dissociation in Protein Quantitative Proteomics ResearchTandem Mass Spectrometry for peptide sequencing is the core technology in large-scale identification of proteins. Collision induced dissociation (CID) is the most commonly used technology for peptide fragmentation mass spectrometry which mainly used in the identification of double charged short peptides. However, due to high energy transfer, the peptide PTM information is often lost in the collision. Relative to CID, in 2004 year, the new electron transfer dissociation (ETD) technology is a gentler and more suitable technology for identifying multi-charged long peptides and keeps peptides PTM information. Restricted to energy transfer efficiency, production of doubly charged ion fragments and the sequence coverage is limited. Since over 70%of trypsin digestion peptides are doubly charged in ESI, the efficiency of ETD fragmentation is quite low. Currently, the researchers mainly increase the efficiency of ETD fragmentation by raising the charge state of peptides including 1. Increase the temperature of sheath gas; 2.increase the surface tension of water mobile phase; 3. Digest with AspN, LysC to produce large peptides; 4. Chemically modify the peptide to make it more suitable to carry more charges. To compare these methods in improving the efficiency of the performance of ETD identification, we used to LysC to produce larger peptides, guanidylation of the tryptic peptides with dual-methylation modification and adding low-volatile-nitrotoluene (m-NBA) which has high surface tension to the mobile phase of chromatography to assess the identification of SILAC labeled AMJ2 cell line in LC-ETD-MS experiment. This thesis for the first time applied mNBA. dual-methylation and guanidine reaction to the analysis of complex biological samples, and observed increased charge state in all ETD-MS based quantitative analysis which facilitated the quantification analysis based on ETD-MS.