Mass Spectrometry based Analytical Methods for Proteome Analysis and Redoxomics

Proteomics is nowadays one of the most important research topics in life science for understanding many life activities from the molecular level. The objective is to find out proteins or modifications on proteins that regulate biological events, aging, diseases and so forth. Because of the complexity in the structure of proteins, the dynamic range of protein-abundances, and the differences from time-to-time and tissue-to-tissue, proteomics requires development of analytical methods with respect to resolution, sensitivity, speed, etc. In this thesis, analytical methods based on mass spectrometry are developed for proteome research and especially redox proteomics. Two major types of mass spectrometers are employed, including matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS) and electrospray ionization mass spectrometer (ESI-MS). Nano and micro metal oxide materials are used to modify the MALDI sample plate for in-situ extraction of phosphorylated peptides or for in-source photochemical reactions. The former is valuable for the study of protein posttranslational modifications. The latter results in online tagging of cysteine for counting the number of cysteine in peptides that is valuable information for the identification of cysteine-containing proteins. Functional microfluidic chips are designed as microreactor and ESI emitter for online chemical reactions followed with MS characterization. Specifically, the multi-channel microchip is applied in tyrosine nitration study. Tyrosine nitration is considered as a biomarker for radical associated diseases, such as cardiovascular, Alzheimer’s, and Parkinson’s diseases. With the microchip ESI-MS, a new pathway for tyrosine nitration induced by copper(II) is demonstrated. Considering that abnormally high concentration of copper(II) was found in the brain tissue of patient with Alzheimer’s disease, the current finding is important for understanding the mechanism of neurodegenerative diseases. An electrostatic-spray ionization (ESTASI) method is developed to generate ions from sample solutions. The ESTASI can be realized with various geometries, such as samples in a microchannel, in a capillary, in a micropipette and on a plastic plate. Comparing to ESI, voltage is not directly applied on the sample during ESTASI, thereby avoiding redox reactions during ionization. The ESTASI has been used to couple separation strategies with MS, including capillary electrophoresis and gel isoelectric focusing. These new methods can be useful in top-down proteomics and mass imaging.