| The onus of providing a highly pure biological sample for mass spectrometric analysis is daunting. For the predominant types of analytes such as proteins, peptides, and nucleic acids, their polymeric nature inherently implies only small differences in their physical properties to draw upon in developing analytical strategies.;In addition, we examine methods of producing a thinner polyurethane surface to reduce space charge effects in a linear TOF system. The formation of thin, poreless polyurethane films was evaluated, as well as the effects of solvent composition on surface artifacts.;The performance of a prototype MALDI-QqTOF was evaluated for the analysis of multiply-phosphorylated peptides. Analysis of the tryptic fragment T1-2 of beta-casein was performed, showing in-source fragmentation of the phosphate moieties. Further MS/MS analysis of the fragment shows the formation of dehydroalanine residues from phosphoserine. In addition, the formation of multiple ion series arising from differing fragmentation pathways of phosphoserine is also seen. Further MS/MS studies of prompt decay products shows similar results, concluding that phosphoserine fragmentation yields multiple ion series even if all phosphoserine have been reduced to dehydroalanine by in-source decay prior to MS/MS analysis.;The evaluation of an on-target electrophoresis system was evaluated for the separation of peptide standards. Samples were electrophoretically separated in a Teflon channel, immobilized by freezing, and lyophilized to remove water prior to deposition of matrix and analysis by MALDI-TOF. Results show separation occurring, though sensitivity is low. Though the Teflon channel allows for electrophoretic separation, it reduces sensitivity in direct MALDI analysis of components, as well as problems in matrix deposition.;Our research focuses on the development of novel analytical tools for the purification of biological samples to be analyzed by mass spectrometry. First, we evaluate surface adsorption of proteins to polyurethane substrates to capture target molecules. Samples of beta-casein are adsorbed onto a polyether type polyurethane surface. Wash steps, and the evaluation of on-membrane digestion parameters are performed to yield peptides suitable for mass spectrometric analysis. In comparison, online CE-MS is evaluated to determine its efficacy in yielding complete peptide maps of phosphorylated peptides. |
