Date of Award

2011

Degree Type

Dissertation

Department

Chemistry

First Advisor

Anderson, David

Subject Headings

High performance liquid chromatography, Mass spectrometry, Ion exchange chromatography, Proteins -- Analysis, HPLC, LC-MS, protein analysis

Abstract

Gradient chromatofocusing-mass spectrometry is a new technique for protein analysis recently introduced by our research group. Capable of separating and identifying proteins according to pI values and molecular weight, gradient chromatofocusing-mass spectrometry has been achieved by integrating a new ion-exchange chromatography technique called gradient chromatofocusing with a newly discovered buffer system that promotes mass spectrometry detection. Differing from traditional ion-exchange chromatography techniques, gradient chromatofocusing employs specific low molecular weight, volatile buffer components that are introduced onto an ion-exchange HPLC column by programming a binary gradient pumping system to deliver the correct proportions of acidic mobile phase to overcome buffering of the column's stationary phase initially equilibrated with a basic mobile phase thus creating a linear pH gradient through the column. Offering greater control of the slope of the pH gradient and improving separation capabilities through usage of buffers at higher concentrations, gradient chromatofocusing buffer systems offer compatibility with mass spectrometry detection that is not possible using polyampholyte buffers commonly used with traditional ion-exchange chromatography techniques. This compatibility led to the first reporting of ion-exchange chromatography being interfaced with mass spectrometry by a previous group member who used a 2.1 mm i.d DEAE weak anion-exchange column and a 25 mM buffer system consisting of ammonium bicarbonate, pyridine, lactic acid and acetic acid. Furthermore, the focus of this dissertation will be to develop an optimized capillary gradient chromatofocusing-mass spectrometry system (Chapter 4) capable of detecting at the low-levels associated with proteomics by miniaturizing the HPLC system (Chapter 2) and effectively operating with the lowest buffer concentrations possible to generate linear pH gradients to promote compatibility with the mass spectrometer (Chapter 3). Similar to capillary gradie

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