Development of on -line chromatographic separations/mass spectrometry and applications in protein analysis.

On-line sheathless capillary electrophoresis-tandem mass spectrometry with stored waveform inversed Fourier transform (SWIFT) isolation technology in an ion trap/reflectron time-of-flight mass spectrometer has been developed with sufficient speed to maintain the CE separation and provide optimized MS/MS spectra, on-line, of each peak. The ion trap performs ion accumulation, ion isolation (with SWIFT waveforms), ion resonant excitation (with TICKLE waveforms) and ion ejection for TOF analysis. The CE/MS/MS system can provide high-resolution separations of complex mixtures and detailed structural analysis at very low levels. In order to improve the speed and sensitivity of protein structural characterization in proteomics, on-line CE/MS has been used to analyze the proteins separated from two-dimensional gel electrophoresis. To circumvent the two conventional problems in the CE analysis: concentration limit of detection and sample clean up before injection, ZipTip C18 was used for preconcentration and purification of sample. This off-line purification method maintains the high resolution of the CE separation where protein digests can be detected from femtomole levels of material injected and a relatively broad coverage of the protein sequence can be obtained. The combination of MALDI/MS, LC/MS and CE/MS/MS provides a systematic and comprehensive method for protein structural analysis including primary structure and posttranslational modifications. The present focus involves a study of myelin basic protein (MBP) isoforms to understand the evolution of this protein in different organisms over the last four hundred million years. We have studied dogfish MBP where our work has clearly demonstrated that residue 30 should be lysine instead of glutamine in the reported sequence. Exon 5 was found missing in C3 by MALDI and CE/MS/MS after CNBr and trypsin digestion. Multiple phosphorylations were present in dogfish MBP isoforms. Other modifications such as deamidation and citrulline were also found in the dogfish MBPs.Ph.D.Analytical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132600/2/9977184.pd