Date of Award


Degree Name

Doctor of Philosophy



First Advisor

Kinsel, Gary


The focus of this dissertation is the synthesis, development and application of polymer brushes for the fractionation of complex mixtures of peptides and digested proteins prior to MALDI-MS analysis. Even though MALDI-MS analysis is increasingly used in proteomic applications, this method of analysis suffers loss of performance in the analysis of mixtures due to the ion suppression effect i.e. basic peptides are more easily ionized and suppress the ionization of other less basic peptides. In the approach taken a polyelectrolytic polymer brush containing randomly polymerized NIPAAM-co-MAA was utilized as a mixture fractionation substrate. This polymer brush was chosen because the long polymer chains are expected to offer a three dimensional volume for adsorption of targeted analytes. In this work fractionation occurs as a result of electrostatic attraction or repulsion between the negatively charged polymer surface and positively or negatively charged analytes in solution, respectively. Fractionation studies utilizing this new method of separation were done first with binary peptide mixtures. Basic peptides are observed to bind to the anionic brush polymer surface, allowing the suppressed acidic peptides to appear in the mass spectra of the wash solutions. Following this successful demonstration, more complex mixtures of peptides derived from enzymatic digestion of proteins were also studied. After fractionation, MALDI mass spectra of both the washed and eluted fractions are obtained and ion signals are analyzed and associated with predicted peptide sequences. Complete analysis of the ion signals using PeptideMap leads to a percent protein sequence coverage. Comparison of this number with the percent protein sequence coverage obtained from unfractionated digest peptide MALDI mass spectra reveals that fractionation significantly increases the information content of the mass spectra. The performance of this polymer brush surface was also optimized. Temporal studies confirmed that a period of three minutes was sufficient for the adsorption of the peptide analyte. This time is required for the polymer brush to expand and collapse to capture and release peptides attracted to the polymer brush surface. In addition to the time required for fractionation, the solvent environment needed for efficient fractionation was also studies. Consistent with expectation analyses indicated that the most effective fractionation occurred at intermediate pH values, while poor separation occurred when fractionation was carried out at higher or lower pH. Lastly, to probe the potential greater capacity of the anionic polymer brush for uptake of peptides, two approaches were employed to alter the structure of the polymer brush surface. One approach was to vary the proportion of MAA to NIPAAM and the other was to increase the photopolymerization time. While the investigations require additional work, preliminary results demonstrate that higher capacity for peptide binding is achieved at longer photopolymerization times. However, the results also suggest that a much more aggressive method is needed to release the peptides bound on the surface of this type of polymer brush.




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