Qualitative analysis of proteomes with isotope labeling
Abstract
This study demonstrates a heavy isotope coding strategy for the analysis of all types of tryptic peptides, including those that are N-terminally blocked and from the C-terminus of proteins. The method exploits differential derivatization of amine and carboxyl groups generated during proteolysis as a means of coding. The technique of tagging amino and carboxyl termini (TACT) at peptide termini is a truly global isotope coding scheme. TACT was used to recognize single amino acid polymorphism between proteins from different sources. After double labeling, all the peptides having the same sequence in the two proteins will appear as a doublet in mass spectra. Uniquely different peptides coming only from one protein will appear as a singlet in mass spectra. TACT recognized six amino acid variations among the variations at seven positions between lysozymes from chicken and turkey. Amino acid variations of canine serum albumin were identified at position 359 and 474. The second portion of the work is focused on identification of disulfide linked peptides with 18O labeling. Two peptides linked by the disulfide bond have two C-termini. Each C-terminus will incorporate two 18O atoms in the 95% 18O enriched water with the presence of trypsin. Therefore, the disulfide linked peptides containing two C-termini will be labeled with four 18O atoms and will have a unique mass shift of 8 amu, which is used for identification of disulfide linked peptides. The third portion is to identify post-translational modification by rechromatography. Diagonal chromatography is the process of using the same chromatographic separation in two dimensions. A chemical modification such as dephosphorylation was applied to all fractions. The dephosphorylated peptide will have a retention time shift due to the change of hydrophobicity, other peptides will have the same retention time between the two dimensions. The serine, threonine and tyrosine phosphorylated peptides were identified by diagonal chromatography.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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