Multiplexed global internal standard technique (mGIST) using a quaternary amine coding agent

Cathy S Sioma, Purdue University

Abstract

The goal of this research was to investigate the use of a quaternary amine coding agent for the use in Multiplexed Global Internal Standard Technology (mGIST). The work reported here began with the search for a reagent that would improve on the limitations of current GIST reagents. Literature reviews and initial studies indicated that a quaternary amine coding agent would best fit the requirements. Based on the success of N-hydroxysuccinimide (NHS) activated acylating agents, the NHS ester of 4-trimethylammonium butyrate (NHS-TMAB) was chosen for these studies. This was followed by work that focused on sensitivity of derivatized peptides in MALDI-TOF-MS when labeling was achieved through TMAB derivatization and to show that TMAB derivatized peptides had an increased sensitivity compared to peptides coded with other GIST reagents. Comparisons of sensitivity were also made between lysine and arginine containing peptides derivatized with the TMAB coding agent. Other studies showed that the TMAB coding agent minimizes the chromatographic isotope effect, creating an ideal internal standard for all tryptic peptides in a biological extract. Finally, the efficacy of isotopic isoforms of TMAB coding reagents was examined in sample multiplexing experiments directed at simultaneous analysis of multiple samples. With the multiplexed GIST approach, the identification of proteins varying between two or more regulatory states became not only possible, but also an effective method to study regulatory flux in a single experiment.

Degree

Ph.D.

Advisors

Regnier, Purdue University.

Subject Area

Analytical chemistry

COinS