Application Of ¹⁸ O Combined With Dimethyl - Labeled Secondary Mass Spectrometry In The Study Of Hepatocellular Proteome

Recently, with the rapid development of proteomics, quantitative proteomics, one of the most important components of proteomics, has been paid more and more attention. With the development of multidimensional liquid chromatography (MDLC) and mass spectrometry (MS), MS-based quantitative proteomics has become mainstay in this field, and various MS-based quantitative methods were developed and widely applied in biological and medical research. However, these quantitative methods have their inherent limitations. So, based on these limitations and specific research requirements, establishing new quantitative methods is still one of the hotspots in quantitative proteomics. In this work, we developed a method, named isobaric MS2quantification by coupling18O labeling and dimethylation (IMS2Q[18O+DM), and applied it to profile quantitative proteome of hepatocellular carcinoma (HCC) liver tissues and adjacent non-tumor liver tissues. Differentially expressed proteins in HCC liver tissues will provide important information for next targeted investigations of potential HCC biomarkers.This dissertation consists of4parts and the contents are summarized as follows:In the first chapter, the advances in the research of quantitative proteomics were summarized. Furthermore, a review of quantitative proteomics study of HCC was presented. Lastly, the aims and significance of this dissertation were presented.In the second chapter, the method of isobaric MS2quantification by coupling18O labeling and dimethylation (IMS2Q[18O+DM]) was developed by standard proteins, and its utility of complex samples was tested by rat liver. In general, trypsin-digested peptides of two samples were separately labeled two16O or18O atoms at their C-termini in H216O or H218O, resulting in a4Da mass difference to the same peptides of different samples. After guanidination, the16O-labeled peptides were dimethylated with CD2O and the18O-labeled peptides were dimethylated with CH2O. These isobaric labeled peptides were indistinguishable in MS1spectra and produced b, y fragment ion pairs in MS2spectra. Multiple b, y fragment ion pairs dramastically improved the peptide and protein quantification accuracy. Three standard proteins were used to demonstrate the accuracy, reproducibility, dynamic range, and isotope effect of IMS2Q[18O+DM], the results showed that the method had a good accuracy and reproducibility, as well as a good linearity within10fold dynamic range with R2>0.99. Besides, no chromatographic shift of the isobaric labeled peptides was detected in RPLC. The utility of IMS2Q[18O+DM] to rat liver samples showed that this method had a similar good accuracy and reproducibility for complex samples.In the third chapter, IMS2Q[18O+DM] was applied to profile quantitative proteome of HCC liver tissues and adjacent non-tumor liver tissues. A total of124proteins were significantly changed in HCC liver tissues, among which45proteins were up-regulated and79proteins were down-regulated. Quantitative results of many proteins were consistent with the previous studies, indicating IMS2Q[18O+DM] can be used for large-scale analysis of biological samples with good accuracy and reliability.In the last chapter, a summary was made and a prospect was presented.