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Analytical and computational approaches for quantitative phosphoproteomics / by Jason M. Gilmore.

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Author	Gilmore, Jason M.
Title	Analytical and computational approaches for quantitative phosphoproteomics / by Jason M. Gilmore.
Date	2014
Abstract	Protein phosphorylation is an important regulatory mechanism for the control of countless processes in living systems. The delicate management of phosphate attachment to, and removal from, serine, threonine and tyrosine residues in proteins regulates progress through the cell cycle, protein subcellular localization, protein complex formation, and protein degradation pathways among many other functional roles. Conversely, disregulation of phosphorylation is frequently associated with disease states and is a hallmark of cancer. Therefore, the comprehensive quantification of protein abundance as well as protein phosphorylation in complex biological samples would provide critical insights into the disregulated cellular processes of cancer and other diseases. However, such analyses are complicated by sample complexity, stoichiometric limitations, and technical challenges associated with the accurate quantification of phosphorylated proteins. Contemporary strategies utilize isotopically labeled heavy standards as controls for tandem mass spectrometry analysis which allow relative quantification between tissue and tumor samples. Here, I describe technical and computational modifications to existing phosphoproteomic workflows which address current limitations of quantitative coverage. Through novel application of protease treatments before tandem mass spectrometry analysis, underexplored regions of the human phosphoproteome become accessible. Additionally, I have developed a generalized extension to the spike-in standard workflow coupled to a computational quantification strategy for the analysis of tissue samples. This method recapitulates the high precision quantifications obtained by traditional techniques and provides novel information for a substantially greater number of peptides and proteins. Finally, I evaluate the prevailing theories concerning lower identification efficiency in phosphoproteomic analyses. This includes challenges for confidently identifying the more complicated spectra of phosphorylated peptides as well as hydrophobicity dependent changes to chromatographic retention, undesirable charge shifts and reduction in ionization efficiency. Ultimately, I find that reduced sensitivity suggests the need for more sophisticated MS/MS ion selection and more advanced label-free quantification methods
Description	viii, 157 leaves : ill. (some col.)
Format	application/pdf
College	Dartmouth Medical School. Department of Genetics.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.1228

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