Enhancing shotgun proteomics by mass defect labeling of tryptophan, improving mass measurement accuracy and on-target digestion
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Proteomics is the large-scale study of the proteins expressed in a cell, tissue and organism under a given set of physiological or environmental conditions. Mass spectrometry (MS) in combination with a variety of separation methods has become a primary tool for proteomic studies. Of all proteomic approaches, shotgun proteomics has gained tremendous popularity in recent years. This method involves batch proteolysis of a protein mixture, separation of peptides by high performance liquid chromatography (HPLC), and protein identification from mass spectrometric analysis of peptides. This dissertation describes three approaches to facilitate shotgun proteomic analysis by accurate mass measurement using Fourier transform ion cyclotron resonance mass spectrometry (FTICR/MS). The first approach, referred to as mass defect labeling (MDL), is based on the derivatization of tryptophan residues in protein with a reagent that alters their mass defects. We describe here the development and application of a tryptophan MDL reagent that is compatible to matrix-assisted laser desorption/ionization (MALDI). The second approach employs the stored waveform inverse Fourier transform (SWIFT) excitation to improve the mass measurement accuracy for higher mass peptides. We demonstrate that sub part-per-million (ppm) can be achieved by combining SWIFT with stepwise-external calibration. The third approach couples accurate mass measurement with nitrogen stoichiometry to improve protein identification in shotgun proteomics using HPLC-MALDI-FTICR/MS. We present here the results from identification and quantitation of the proteins in a 15N-metabolically labeled proteome sample that was obtained from Methanococcus maripaludis. The forth approach combines on-target digestion with MALDI-FTICR/MS analysis for rapid protein identification. On-target digestion of a HPLC separated proteome with MALDI-FTICR/MS analysis offers an alternative to the standard method of shotgun analysis.