Method Development for Mass Spectrometry-based Bottom-up Proteomics Using Nonenzymatic Digestions

High throughput protein identification can be achieved in a mass spectrometry-based bottom-up proteomics workflow. In a typical process, a proteome of the target organism is usually first digested into short peptides by trypsin, an enzyme which specifically cleaves at the C-terminus of the arginine and lysine residue in the protein sequence. The mixture of peptides is then separated by reverse-phase liquid chromatography (RPLC), and a mass spectrometry (MS) is coupled downstream. As the peptides elute from the RPLC, the MS performs online measurement of the mass-to-charge (m/z) of the ionized peptides and their amino acid sequences can also be acquired through fragmentation inside the MS. In parallel with the experimental process, an in silico digestion is performed on the proteome database of the target organism. Because the production of the species of peptides from any protein is predictable, by matching the experimental data and the in silico digestion proteolytic peptides, proteins can be identified.;However, the use of trypsin requires prolonged digestion time (2 to 18 hours), limiting the speed of analysis. Non-enzymatic digestions as alternatives offer great potential in terms of digestion speed and robustness. In this dissertation, thermal decomposition digestion (TDD, cleavage occurs at the C-terminus of aspartic acid and N-terminus of cysteine), microwave D-cleavage (cleaves at the C-terminus of aspartic acid) and electrochemical oxidation (C-terminus of tryptophan and tyrosine) were investigated for their application in bottom-up proteomics. TDD on phosphorylated peptides introduced neutral loss of the phosphate moiety, resulting -80 Da and -98 Da on the phosphorylated residue. In addition, TDD also caused site-specific cleavage on N-Calpha bond of phosphorylated serine residue through beta-elimination and successive gamma-hydrogen elimination. These results suggested that TDD is better suited for protein and peptide sequence study rather than acquiring information of post-translational modifications. While TDD offered a digestion method for solid samples, microwave D-cleavage and electrochemical oxidation were performed in solution. The combination of microwave D-cleavage and electrochemical oxidation is of special interest with its potential to overcome individual shortcomings stemming from each digestion method; moreover, the utilization of two digestion methods allows the implementation of a strategy to perform two-dimensional separation through sample digestion. This strategy added an addition RPLC between the first and second digestion method, exploiting the changes of the hydrophobicity of the sample caused by the second digestion. The retention time of the sample was different before and after the second digestion and only one separation technique (RPLC) was needed to achieve two-dimensional separation. In silico analysis on the E.coli. proteome showed that this strategy was able to achieve sample orthogonality with R2 value (coefficient of determination, scaled from -1 to 1, where -1 stands for 100% orthogonality and 1 for 0% orthogonality) from -0.73 to 0.99, depending on the choice of digestion methods. In order to implement this strategy to the microwave D-cleavage and electrochemical oxidation, several liquid chromatography system configurations were designed and tested using peptide standards. The results showed it was crucial to maintain optimal sample concentration for both effective electrochemical oxidation and RPLC separation resolution.