The development of highly selective and very sensitive methods to detect peptides and proteins of interest in complex mixtures remains an important goal in proteomics applications. This dissertation focuses on the use of reverse-micelle forming amphiphilic homopolymers as part of liquid-liquid extraction to selectively extract and concentrate peptides from an aqueous solution into an immiscible organic phase. After extraction, the polymer-peptide mixtures are amenable to direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). The charged interiors of the reverse micelles enable oppositely charged peptides to be selectively extracted into the aggregate’s cores via coulombic attraction. Reverse micelles formed by negatively charged carboxylic acid or positively charged quaternary amine groups can be used alone or in sequence to selectively extract and fractionate peptides according to their isoelectric points (pIs). Furthermore, the pI cutoff can be readily tuned by adjusting the extraction pH. The coalescence of polymer-peptide conjugates into hotspots on the MALDI target plate during MALDI-MS analysis results in significant signal enhancement for the enriched peptides, enabling reproducible ion signals at concentrations as low as 10 fM. Interestingly, reverse micelles formed by positively charged polymers with quaternary amine substituents can selectively enrich acidic peptides that are undetectable during regular MALDI-MS analysis. The extraction protocol along with MALDI-MS can also be used for the selective enrichment and detection of low abundance peptide/protein biomarkers in human serum at physiologically relevant concentrations. Overall, the results described in this dissertation reveal that this selective extraction protocol along with MALDI-MS analysis might have a significant impact on protein identification and early stage screening of biomarkers.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-6475 |
| Date | 01 January 2011 |
| Creators | Rodthongkum, Nadnudda |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
Page generated in 0.0159 seconds