| Two of the major objectives of protein biochemistry are the identification of protein post-translational modifications and the elucidation of relative protein expression in dissimilar cell types. Mass spectrometry (MS), possessing both high sensitivity and great specificity, has emerged as the technique of choice to address these goals. In the three biological systems presented here, the technique of nano-flow RP-HPLC micro-electrospray ionization (nHPLC-muESI) MS has been applied to peptide sequencing, protein identification, and differential analysis.; In the first study, nHPLC-muESI MS on a quadrupole ion trap instrument was used to identify phosphorylation sites in the Upstream Binding Factor (UBF), a protein that plays a critical role in the regulation of protein synthesis and cellular growth. Hyperphosphorylation of UBF initiates the transcription of ribosomal RNA (rRNA), the rate-limiting step in ribosome biosynthesis. Proteolysis of nuclear-extract treated, recombinant UBF, followed by immobilized metal affinity chromatography (IMAC) for phosphopeptide enrichment, and analysis on a quadrupole ion trap mass spectrometer led to the identification of several novel phosphorylation sites.; In the second study, nHPLC-muESI MS on both a quadrupole ion trap instrument and a Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometer was used to identify those proteins exhibiting differential expression in anaerobically versus aerobically grown Pseudomonas aeruginosa. These bacteria flourish within the hypoxic environment found in the mucus lining the lungs of Cystic Fibrosis patients and are the eventual cause of mortality in most cases. Several of the proteins identified using this technology represent novel targets for the treatment of chronic P. aeruginosa infections, as humans lack the enzymatic machinery necessary for anaerobic sustenance.; In the third study, nHPLC-muESI MS on a hybrid linear ion trap/FT-ICR mass spectrometer was used to investigate the differential phosphorylation of proteins during capacitation, the process by which sperm cells acquire the ability to fertilize an egg. Proteolysis of total protein extracts from both capacitated and non-capacitated sperm, followed by differential isotopic labeling of the resulting peptides, MAC enrichment, and analysis on a hybrid linear ion trap/FT-ICR mass spectrometer, permitted the global quantitation of phosphorylated peptides and was used to identify a number of phosphorylation sites induced by capacitation. |
