Development And Application Of New Mass Spectrometry-based Proteomics Technologies To Post-translational Modifications

Abstract

Post-translational modifications (PTMs) represent one of the major cellular mechanisms to diversify the limited number of proteins coded by genome. They provide dynamic regulation of protein functions and the means to fine-tune protein functions in response to the changing cellular environment and physiological conditions. Despite their crucial roles in cellular functions, efficient and sensitive analysis of PTMs remains a daunting analytical challenge. Traditional chemical approaches to characterize PTMs are labor-intensive and time-consuming, often with low sensitivity and specificity. Over the past ten years, biological mass spectrometry has emerged as an indispensable tool to identify and characterize PTMs. Mass spectrometry-based proteomic study of PTMs has become possible with the development of new analytical instruments, chromatographic technology, new techniques for enriching peptides bearing a PTM, and bioinformatics software. However, the reliability of PTM identifications with current technologies remains questionable, hindering the process of further understanding the biological functions of PTMs. Therefore, new mass spectrometry-based technologies are much needed to improve the sensitivity and reliability of PTM identification. Towards this end, my thesis describes the development of new mass spectrometry-based proteomics technologies for PTMs. It begins by introducing strategies for systematic manual verification of tandem mass spectra for peptide and PTM identification as well as the identification of common types of false positive sequence alignments from representative bioinformatics computer programs. Our results challenge some of the most popular concepts in the analysis of tandem mass spectrometry data and point out the caveats in the common practice of sequence alignment for peptide identification. The application of the strategies and concepts to manual verification led to the successful discovery of two novel protein modifications: lysine propionylation and lysine butyrylation. Our results demonstrated that these two novel modifications are dynamic PTMs that can be regulated by enzymes in a manner similar to lysine acetylation. We further developed a new software tool called PTMap based on the concepts and strategies of manual verification for unrestrictive PTM identification. The software was demonstrated to have high accuracy and efficiency compared to other known sequence alignment tools. The application of the software to the analysis of four selected proteins, human histone H4, HMG2, mouse SGK1 and BSA, led to the identification over seventy novel PTMs and polymorphisms. The data suggest that the complexity of the proteome exceeds far beyond what people have imagined. In another demonstration, the application of the software led to the discovery of four novel chemical modifications introduced mainly during in vitro sample handling, providing guidance for better sample preparation. Together, the strategies and tools described in this thesis provide a powerful platform to fully dissect post-translational modifications using mass spectrometry and explore their dynamic implications in biological processes.