Derivatization strategies as an aid to assist separation and mass spectral properties of peptides in proteomics
Abstract
In the past few years, derivatization based strategies have dominated the wide spread effort of differential chemical analysis of biological systems, especially in the field of proteomics. Derivatization strategies have been used to quantify changes in protein expression, to simplify complex protein mixtures and to enhance representation of low abundance proteins. In this thesis, two new derivatizing strategies were developed and validated. Sulfo-succinimidyl benzoate, a new Global Internal Standard Technology (GIST) reagent, quantitatively derivatizes primary amine groups of peptides. The reagent can be developed as a 13C isoform, thereby eliminating any chromatographic resolution issues between coded peptide pairs. The hydrophobicity accrued after benzoyl derivatization assists in retention of short peptides often lost in reversed phase chromatography. Benzoyl derivatization is especially useful for enhanced coverage in tryptic mapping, detection of single amino acid polymorphism and retention of even single amino acids during reversed phase chromatography. An alternative residue specific strategy was also developed. The derivatization strategy selectively targets only cysteinyl peptides from complex tryptic digests using a quaternary amine reagent. This increases the positive charge on most cysteine-containing peptides to the point that they can be selected with a SCX column, thereby assisting in sample simplification. Simultaneous quantification and selection of cysteinyl peptides was achieved using isoforms of 3-acrylamidopropyl trimethylammonium iodide. The effect of different C-termini amino acids on chromatographic resolution of 16O/18O coded peptide pairs during reversed phase chromatography was also investigated. In a comparative study, it was observed that no significant resolution is observed for peptides cleaved using different proteases.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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