Date of Award

12-1-2014

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Kinsel, Gary

Abstract

AN ABSTRACT OF THE DISSERATION OF STEPHANIE EASTWOOD, for the Doctor of Philosophy degree in CHEMISTRY, presented on the DATE OF DEFENSE, at Southern Illinois University Carbondale. TITLE: DEVELOPMENT OF AMINE CONTAINING POLYMERIC MODIFIED MALDI TARGET SUBSTRATES FOR COMPLEX MIXTURE FRACTIONATION OF PEPTIDE AND PROTEIN MIXTURES PRIOR TO MALDI MASS SPECTROMETRY ANALYSIS MAJOR PROFESSOR: Dr. Gary Kinsel The focus of this dissertation is the synthesis, development and application of the amine containing modified targets for the fractionation of complex mixtures of peptides and digested proteins prior to MALDI-MS analysis. Even though MALDI-MS is increasingly used in proteomic applications and analysis, this method suffers from loss of performance in the analysis of mixtures, due to the ion suppression effect. In this effect, basic peptides ionize more readily than acidic peptides and there by suppress the ionization and detection of less basic peptides resulting in the loss in the detection of valuable sequence information and posttranslational modifications. In the approach taken, a modified target containing amine functionality was utilized as a mixture fractionation substrate. The substrates were chosen for the adsorption of targeted analytes. Incorporating this amine function into 3D brushes offer the ability to absorb higher quantities of peptides, allowing for a more thorough analysis and more sequence coverage. In this work fractionation occurs as the result of electrostatic attraction or repulsion between the positively charged polymer surface and negatively charged analytes in solution. Fractionation studies utilizing this amine chemistry for separation were done first with binary peptide mixtures. Basic peptides are observed to repel from the cationic polymer surfaces, allowing the suppressed acidic peptides to separate from the mixture by binding to the amine substrates. The acidic peptides can be eluted and observed. Following successful demonstrations, more complex mixtures of peptides derived from enzymatic tryptic digestion of proteins were studied. Following fractionation, MALDI mass spectra of both the washed and eluted fractions are obtained and ions signals are analyzed and associated with predicted peptide sequences. Complete analysis of the ion signals using Prowl and PeptideMap leads to a percent protein sequence coverage. Comparison of this number with the percent sequence obtained from unfractionated (conventional) digested peptide mixture mass spectra reveals that fractionation does in fact significantly increase and also increases discovery of post translational modifications (PTM). Fractionation studies were studied by varying the pH of tryptically digest proteins. Sequence coverages were found to increase upon fractionation at all pHs studied. In order to increase the discovery of PTMs, the cationic polymer brush was doped with copper ions to selectively capture phosphopeptides. Negatively charged phosphopeptides were shown to be selectively captured by the copper ions complexes within the polymer brush substrate, and subsequently released upon addition of acid.

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