A Novel Method Of Capillary Electrical Heating Apparatus And Arginine Labeling In Capillary HPLC-MS Quantitation

With comprehensive proteomics research and the development of proteomics technologies, the proteomics focus has turned from qualitative research such as protein identification, post-translational modification and protein-protein interaction to quantitative studies including differential proteomes between different biological states or healthy and pathological states as well as the absolute determination of some key proteins. Accurate quantitation methods for a target proteome have become the current research hot point and their development faces many challenges. To address the problems in quantitative proteomics, such as highly efficient separation of a complex biological sample and deep coverage of proteins with low abundance identified and quantified, a longer capillary chromatographic column or a column packed with packing materials with smaller particle size is employed, but the column back pressure rises notably which often causes liquid leak at connective components. Therefore, in my thesis, a simple electrical heating apparatus was first designed and made, and its performance evaluated. Then, in order to determine the absolute quantities of proteins in a biological sample with high accuracy and sensitivity, a novel absolute quantitation method using arginine labeling combined with multiple reaction monitoring mass spectrometry (MRM MS) was developed.This thesis consists of three chapters. In the first chapter, an introduction to recent quantitative proteomics, the importance of HPLC separation and its present progress and problems were first described. Then, the current advance of labeling quantitation in proteomics, and the significance of the absolute quantitation of drug metabolizing enzymes were also reviewed.In the second chapter, a simple method of making an electrical heating apparatus for raising the temperature of capillary columns and its application to LC-MS system was proposed, and its performance was evaluated with bovine serum albumin (BSA) tryptic digest and the tryptic digest of yeast proteins in terms of column pressure and column efficiency. The results showed that at the optimum current, our electric heating apparatus could reduce the column pressure of a capillary column packed with 3 μm packing materials by at least 50% compared with the pressure without electric current applied in the apparatus during the separation of BSA tryptic digest and yeast tryptic digest, and the column efficiency increased slightly. This suggests that the electrical heating apparatus can significantly reduce the column back pressure, which provides us a practical way to use a capillary chromatographic column packed with packing materials with smaller particle size at a lower pressure.In the third chapter, a novel method of arginine labeling combined with MRM MS was established in which arginine was attached to the N-termini of the peptides by Staudinger reaction and the reaction between N-succinimidyl group and the N-termini. The results showed that arginine labeling is highly efficient and meets the demand of the establishment of a quantitation method. Both isotope labeled peptides had the same chromatographic retention times and appeared in pairs with the nearly same peak intensity. The foundation of this method offers an alternative choice for protein and proteome quantitation in complex samples.