Doctor of Philosophy (PhD)
Protein separation by polyacrylamide gel electrophoresis (PAGE) and identification by matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) are primary tools of protein analysis. In these techniques, surfactants are used in protein sample preparation in order to enhance the protein solubility. Conventional surfactants have shown limitations in protein analysis due to the structural complexity of proteomes, resulting in low resolution. The research goal of this dissertation is to address some of these limitations by applying novel cationic ionic liquid surfactants (ILS), N-alkyl-4-methyl pyridinium bromide (CnPBr where n=4, 8, 11). The ILS would be suitable candidates to be used in PAGE protein separations as a result of positive cooperative binding to proteins at low concentrations of ILS and protein denaturing ability at room temperature. These compounds were used as buffer additives in ILS-PAGE protein separation and matrix additives in MALDI-MS protein identification. Anionic ILS-PAGE was used to separate a mixture of acidic proteins by applying ILS in sample and running buffers. Protein separation was improved for transferrin and ovalbumin, which were resolved as multiple bands of isoforms. In cationic ILS-PAGE, ILS were applied in polyacrylamide gels in addition to sample and running buffers. Separation of both acidic and basic proteins as sharp bands with high resolution is a major advancement of this technique. Cationic ILS-PAGE was used to resolve ribonuclease b glycoforms as multiple protein bands. In contrast, the same protein was migrated as a single band in Sodium dodecyl sulfate (SDS)-PAGE. Moreover, alpha antitrypsin glycoforms were resolved as multiple spots by two dimensional (2D) Isoelectric focusing (IEF)/ILS-PAGE. Furthermore, C4PBr and C8PBr ILS were applied as matrix additives with MALDI matrix, α-cyanohydroxycinnamic acid (CHCA), to perform protein sample analysis as well as rat brain tissue profiling. ILS showed high protein signal intensity at low concentrations (0.02% (w/v)) in protein samples compared to SDS, cetyltrimethyl ammonium bromide (CTAB), and no surfactants present (blank). A large number of new protein peaks were acquired from tissue sample as compared to the absence of ILS in the matrix. These results show the applicability of ILS in improved protein identification by MALDI imaging mass spectrometry.
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Vidanapathirana, Punprabhashi, "Improved Protein Characterizations using Ionic Liquids: PAGE and MALDI-MS" (2017). LSU Doctoral Dissertations. 4234.
Warner, Isiah M
Available for download on Saturday, February 23, 2019