A Universal Quantitative Strategy Integrated ¹⁸O Incorporation With MS Multiple Reaction Monitoring And Its Application In HCC Biomarker Research

Biomarker discovery has attached extensive attention in the medical field. The process of biomarker research has been greatly accelerated in recent years by using proteomics methods such as 2-D gel and biological MS. Thousands of biomarker candidates have been discovered by the comparison of the protein expression in normal and disease samples. To obtain reliable biomarkers for clinical use, these biomarker candidates must experience development pipeline including discovery, verification and clinical validation. However, because of the difficulties of antibody development, conventional verification methods have become a bottleneck in the biomarker development. Compared to Western blotting and Enzyme-Linked Immunosorbent Assay (ELISA), SID-MRM-MS can analyze multiple biomarkers in one sample with high specificity without antibody, which showed great superiority on biomarker verification and validation.Although SID-MRM-MS has been accepted in biomarker verification, the broad application of this method in large-scale biomarker verification has been prevented due to the inconvenience and high consumption of stable isotope-labeled peptides as the internal standards for the quantification experiments. The standard Fmoc chemistry method can produce labeled peptides using isotope-incorporated amino acids as a raw material, but these amino acids are expensive and the reaction is time-consuming for large-scale preparation. Alternatively, the method using recombinant protein in medium containing the isotopic amino acids was difficult to control the individual internal standard concentrations to the approximate concentrations of endogenous analytes. Other than the above two methods, chemical modification can also be applied to incorporate an isotopic label into peptides. However, the analysis was inconvenient because endogenous analytes were also labeled.The purpose of this thesis is to establish a universal SID-MRM-MS quantitative method combining ¹⁸O labeled internal standard preparation method with MRM-MS for HCC biomarkers verification in clinical serum samples. This paper consists of 4 parts. In the first chapter, we introduced the principle and characteristics of MRM -MS, the strategy based on MRM-MS and its application in the area of quantitative analysis of post-translational modification protein, protein and RNA interactions and verification of biomarker candidates.In the second chapter, the ¹⁸O stable isotope incorporation was introduced and optimized to prepare internal standard for absolute quantification. The research was focused on the efficiency of ¹⁸O labeling and ¹⁸O-16O back-exchange to optimize reaction conditions and to ensure the reliability of the method. It was the first time to internal standard preparation using this method. First, the labeling efficiency was enhanced by the addition of RapigestTM SF in H2¹⁸O and microwave heating. Second, chemical modification with Tris (2-carboxyethyl) phosphine (TCEP) and iodoacetamide (IAA) resulted in trypsin deactivated completely and no significant back-exchange from ¹⁸O to 16O was observed after labeling in 6 days. Finally,the specificity of MRM detection for ¹⁸O labeled peptide and unlabeled peptides was examined to ensure reliability for absolute quantification. The experiment result showed that the improved method could be effectively used to label peptide and prepare internal standards. The method met the needs of the absolute quantification and provides a new choice for the convenience and high-throughput preparation of the isotope internal standard.HCC biomarker candidates , vitronectin and clusterin, may played an important role in the occurrence and development of cancer. The prior findings indicated that the fluctuation of these proteins in serum was closely related with the liver function impairment. In the third chapter, we developed a fast absolute quantification strategy combined optimized ¹⁸O labeling with MRM-MS to verify two biomarker candidates, vitronectin and clusterin in undepleted serum. In this research, in order to analyze the highly complex serum samples, we optimized MRM condition, serum sample preparation and capillary-HPLC analysis. The reliability of this method was further evaluated by method validation, with accuracy (%RE) and precision (%RSD) of less than 20%, good linearity in the range of 0.4-40 fmol/μL (r2 > 0.99), recovery rate of more than 87% and reproducibility (%RSD) of less than 6.87%.Our quantification strategy was applied in clinical serum samples to verify the concentration of two biomarker candidates vitronectin and clusterin in 20 serum samples including 10 cases of normal serum and 10 cases of HCC serum. The statistical analysis demonstrated the down-regulated of two proteins in HCC group with significant difference. In this study we innovatively established high specificity, high accuracy and reproducible absolute quantification method of proteins combined ¹⁸O labeling with MRM-MS, which will promote wider application of the SID-MRM-MS strategy in the field of biomarker verification.The core fucose glycosylation was an important factor in the development of HCC. But the current techniques, due to complete resection of sugar chains, lost the core fucose glycosylated information and increased false-positive results. In the fourth chapter, we simplified the sugar chain to retain part of the glycosylation information and established the MRM-MS quantitative confirmation strategy coupling with ¹⁸O labeling to verify core fucose glycosylation level of protein biomarkers in serum samples. First, the sugar chain on core fucose glycosylated protein was simplified to retain disaccharide structure by enzymatic digestion. The fragment of simplified fucose glycosylated peptides in triple quadrupole mass spectrometer were analyzed for MRM–MS detection. Then, the relative quantitative strategy of core fucose glycosylated peptide combined ¹⁸O labeling with MRM-MS was developed to verify the six candidate biomarkers in 18 clinical serum samples. Finally, based on the result obtained in the total protein level and the core fucose glycosylated peptide level, the difference of Alpha-2-macroglobulin and Ceruloplasmin in healthy and HCC group was demonstrated. As so far, this is the first method developed to give quantitative verification results focused on the specific glycol site, which provided quantification information of the total protein level and the core fucose glycosylated peptide level simultaneously.