Date of Award

Spring 1-1-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Natalie Ahn

Second Advisor

William Old

Third Advisor

Robin Knight

Fourth Advisor

Michael Stowell

Fifth Advisor

Veronica Bierbaum

Abstract

Mass spectrometry has become the preeminent method in proteomics, making identification of peptides from their pattern of gas phase fragmentation of interest. Current state‐of‐the‐art peptide identification discards intensity information in peptide spectra; however, improved understanding of the gas phase chemistry allows prediction of ion intensities. Improving the prediction of ion intensities requires uncovering novel fragmentation mechanisms and incorporating them algorithmically into programs designed to computationally model spectra. One such mechanism, discussed here, involves unexpectedly avid cleavage when proline is at the second position. Modeling this mechanism indicates that the gas phase chemistry is not catalyzed by charge. No such mechanism has been previously identified. Incorporating the predicted intensities improves peptide identification; however, certain classes of reaction, such as those exhibited in phosphopeptides continue to confound identification.

Reversible protein phosphorylation modulates almost all aspects of cellular function, making understanding of this post‐translational modification essential in modern proteomics. Unfortunately, the phosphoester bond is very reactive, leading to spectra that are convoluted by the neutral loss of H3PO4. By using a dataset tenfold larger than previous studies, I am able to rigorously analyze how sequence affects neutral loss. In contrast to cleavage of peptide bonds, I find that neutral loss of H3PO4 is affected significantly by distal sites, most notably the basic residues and N‐terminus. Previous studies have suggested that basic residues directly catalyze neutral loss and initial analysis shows enhanced neutral loss near bases. However, in an example of Simpson’s paradox, when we stratify the spectra by charge‐mobility, we find evidence for the converse, that nearby bases inhibit neutral loss regardless of mobility class. In mobile proton spectra, the N‐terminus is the strongest predictor of high neutral loss, with proximity to the N‐terminus essential for peptides to exhibit the highest levels of neutral loss. These observations suggest a model in which the phosphate is always in complex with protons immobilized at basic sites though the adoption of secondary structure, in contrast to the prevailing model, which suggests direct protonation of the phosphate. Further evidence suggests that this mechanism may be general to all neutral loss reactions from peptide side chains.

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