| The vision of the proteome of a biological system has been radically refined due to many biochemical and bioanalytical advances in the past decade. In particular, mass spectrometry has had a major impact on understanding these proteomes because of its high sensitivity and accuracy. The enormous complexity of these biological samples demands continuous improvements in dynamic range, sensitivity and specificity to the separation sciences and mass spectrometry. Described here are the studies of two biological systems using nano-flow reverse-phase high performance liquid chromatography coupled with micro-electrospray quadrupole ion trap tandem mass spectrometry (nHPLC-muESI-MS/MS).; In this first study, the proteome of pigment organelle melanosomes at various developmental stages was characterized. Sucrose density gradient fractionation was employed to generate appropriate samples. For the identification of membrane proteins from unpigmented SKmel28 and early stage pigmented MNT1 melanoma cells, protein samples were fractionated by SDS PAGE, digested in gel with trypsin and analyzed by nHPLC-muESI-MS/MS. Analysis of samples from highly pigmented, late stage melanosomes proved to be particularly challenging because many proteins remained associated with or covalently linked to the polar melanin biopolymer. These samples were solubilized in detergent, digested in solution, and melanin was then removed by Fe(III) Immobilized Metal Affinity Chromatography (IMAC) prior to analysis by mass spectrometry. Over 1500 proteins, both common and unique to various stages of melanosome development, will be presented and discussed in the first chapter.; In the second study, the yeast phosphoproteome was characterized with a newly described ion/ion reaction: electron transfer dissociation (ETD) and mass spectrometry. Despite its significance, protein phosphorylation analysis remains challenging. This is in part due to the uninformative phosphopeptide dissociation with conventional collision activated dissociation (CAD) based-MS. ETD, on the other hand, is indifferent to the presence of phosphate and can provide near complete sequence coverage of phosphopeptides. In the second chapter, a unified phosphoproteomics strategy will be described, comprising the following: (a) enzymatic digestion with Endo-Protease-LysC, (b) phosphopeptide enrichment with immobilized-metal-affinity chromatography (MAC), followed by (c) nHPLC-muESI-MS/MS with ETD. Overall, nearly 1100 phosphorylation sites corresponding to over 600 phosphoproteins, having a dynamic range > 25,000, were identified following a 1-day experiment from 30mug of yeast proteins. |
