Isotope coding for qualitative and quantitative study of peptides and proteins: A global internal standard technology in proteomics
Abstract
Proteome research endeavors to examine the total protein complement encoded by a particular genome and to address biological problems that can not be answered by examination of nucleic acid sequence alone. One of the critical analytical issues in proteomics research is to monitor cellular regulation and determine how to quantify up- and down-regulation of specific peptides or proteins. Recognizing concentration changes in biological system involving small numbers of species in large number of similar, fixed components is a major problem. This challenge is exacerbated by the fact that the components in flux are frequently unknown. The objective of this research was to find a quantitative and qualitative approach to study the proteomics. As part of the global internal standard technology (GIST), a method of isotope-coding internal standard was developed based on N-terminal labeling with an acetylating reagent N-acetoxysuccinimide and its isotope analogue. The specificity of the acetylation reaction using N-acetoxysuccinimide as the labeling reagent with different peptide, peptide mixtures and glycan was studied. This work focused on the quantification using this labeling method on both MADLI and ESI mass spectrometry and comparison of the quantification study on these two different mass spectrometric techniques was made. By extending this labeling reaction, a method of using differential isotopic coding to identify the cross-reacting antigens and study the up and down regulation of phosphopeptides in cow milk in both healthy and mastitis-infected state was developed. Finally, an approach of selection of the signature peptide based on N-terminal peptides by an N-hydroxysuccinimide activated silica particle was investigated. Selection of the signature peptide based on N-terminal peptides by an N-hydroxysuccinimide activated silica particle was investigated.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry|Molecular biology
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