Development of an experimental and computational platform for enhanced characterization of modified peptides and proteins in environmental proteomics

Over the last decade, mass spectrometry based proteomics has been established as the front-runner in systems-level protein expression studies. However, with the field progressing into research of more and more complex samples, novel challenges have been raised with respect to efficient protein extraction and computational matching. In this dissertation, various aspects in the proteomics workflow, including experimental and computational approaches, have been developed, optimized and systematically evaluated. In this work, some of the critical factors with respect to proteomics sample preparation, like available biomass, detergent removal methods, and intact protein fractionation to achieve deeper proteome measurements were evaluated. The presented work will help the broader scientific community to carefully design proteomics experiments especially in biomass limited samples.

A second major area of focus in this dissertation is comprehensive characterization of post-translational modifications (PTMs) in different biological systems. PTMs are critical for functioning of both the prokaryotic and eukaryotic species and this dissertation will highlight some of the experimental strategies to explore the diversity of PTMs in microbial isolates via application of alternate protease and multiple fragmentation schemes. The PTM discovery approach will be further extended into a complex eukaryotic model trees species Populus trichocarpa using recently developed sequence tagging methods to carryout broad scale PTM search and a complete blind PTM search.

Although the work presented in this dissertation mainly revolves around prokaryotic and eukaryotic species involved in environmental proteomics, the general considerations outlined in this work can be extended to every proteomics pipeline. Thus this dissertation will benefit the scientific community in carefully designing experiments before embarking on any research project involving mass spectrometry.