The development of novel MALDI and nanoelectrospray mass spectrometry methods for the improved analysis of proteins and peptides
Padliya, Neerav Dilip
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The development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) gave rise to the era of biological mass spectrometry and the new field of proteomics. Two recurring problems that are central to advancing the science of proteomics are the development of reliable mass spectrometry methods to identify proteins and to sequence peptides. The specificity of proteases such as trypsin in combination with the high mass accuracy provided by reflectron time-of-flight mass analyzers, allows the reliable identification of a protein from the mass spectrum of its proteolytic digestion products in an experiment known as peptide mass fingerprinting. Some powerful approaches to improve the confidence of the identification of proteins in the peptide mass fingerprinting experiment have been described in this dissertation. Both approaches described in this dissertation involve the optimization of different aspects of the sample preparation step in the MALDI-TOF mass spectrometry experiment. Precursor-ion scanning in the negative ion mode followed by sequencing in the positive ion mode is a powerful strategy employed to identify and sequence phosphopeptides via nanoelectrospray triple quadrupole mass spectrometry. Less sample and time would be consumed if both of the aforementioned steps could be carried out in the negative ion mode. Nanoelectrospray tandem mass spectrometry in the negative ion mode is usually not successful as traditional gold-coated emitters undergo electrical discharge. Using polyaniline-coated emitters, we have demonstrated that peptides can be sequenced successfully in the negative-ion mode enabling the observation of a wealth of information that is not available in the corresponding positive-ion mode experiment.