Functional proteomics analysis by nHPLC-muESI ion trap mass spectrometry

Functional proteomics is a research field aiming to define the functional roles of proteins involved in living cells. Mass spectrometry has become a powerful tool for functional proteomics analysis due to its high sensitivity. Nano-flow reverse phase-high performance liquid chromatography, micro-electrospray quadrupole ion trap tandem mass spectrometry (nHPLC-μESI-MS/MS) has been applied to the study of three biological systems.; In the first study, two-dimensional liquid chromatography (2D-LC) (strong cation exchange chromatography followed by reverse phase chromatography), coupled with tandem mass spectrometry (2D-LC- MS/MS) was used to obtain a global protein expression profile of E. coli under a single growth condition. A total of 1,147 proteins were detected by mass spectrometry. The protein profile was then compared to its transcription profile obtained by DNA microarray to survey the relationship between protein expression and gene expression for E. coli.; In the second study, the analysis of proteins expressed by early stage melanosome was initiated to understand the biogenesis of melanosomes, the human pigmentation organelle whose dysfunctions can cause many skin diseases including a highly malignant skin cancer melanoma. Two approaches have been tried using either SDS-PAGE followed by reverse phase HPLC-tandem mass spectrometry (1D-LC-MS/MS) or 2D-LC-MS/MS to analyze the melanosomes. Using these approaches, we have identified more than 200 proteins including all 6 of the known melanosomal proteins and several novel proteins whose presence in melanosomes has been confirmed.; The goal of the third study is to map the phosphorylation sites on Rad53 following DNA damage. Rad53, a DNA damage checkpoint protein in budding yeast, becomes activated in response to DNA damage. This activation correlates with its phosphorylation. In this work, purified Rad53 protein was digested with enzymes. The phosphopeptides were enriched by immobilized metal chromatography (IMAC) and analyzed by nHPLC-μESI-MS/MS. A total of 27 unique phosphorylation sites have been identified. The functional role that these sites play in DNA damage checkpoint pathway have been characterized and a new model for Rad53 activation following DNA damage has been proposed.