The analysis of proteins and peptides by mass spectrometry: From the whole cell proteome to small protein complexes.

摘要

Mass spectrometry (MS) is now well established as an extremely powerful analytical tool for protein and peptide analysis, possessing both high-sensitivity and great{09} specificity. It is able to detect signals from as little as 20 attomoles of material. It also possesses the ability to isolate a single species, including post-translational modification on peptides from a background of thousands of co-existing species.; The techniques of nano-flow RP-HPLC, micro-electrospray quadrupole ion trap mass spectrometry, and Fourier transform ion cyclotron resonance mass spectrometry have been applied to the study of three biological systems. In the first, two-dimensional gel electrophoresis was used to resolve protein spots that mediate estradiol-dependent synaptogenesis in the adult, female rat hippocampus. Understanding what molecules underlie the brain's ability to reconfigure itself will hopefully allow intervention when necessary to reactivate these molecular pathways and reconfigure elements of the central nervous system on demand. Proteolysis in situ with trypsin, followed by mass spectrometry, allowed the identification of both known and novel proteins involved in hippocampal synaptogenesis.; In the second study, gel electrophoresis was used to resolve proteins co-immunoprecipitating with the FANCA protein from Hela cell extracts. FANCA plays a critical role in the disease Fanconi anemia and is thought to be involved in a novel mechanism for DNA repair. Mass spectrometry allowed the identification of both known and novel molecular partners of FANCA. A novel site of phosphorylation on FANCG, a FANCA binding partner, was identified and its significance verified biochemically.; In the third study, a methodology is described for identifying proteins from a whole cell proteome involved in the sporulation process of Bacillus subtilis. Fourier transform mass spectrometry was used to generate high-resolution mass spectra and a subtractive analysis was performed to identify proteins that characterize stage II spore formation. Understanding how cell differentiation occurs is a necessary step in elucidating how life develops. Bacillus subtilis provides a simple model to study cell differentiation.; In this dissertation, various types of mass spectrometry have been used for subtractive analysis, peptide sequencing, and protein identification in three independent biological systems, demonstrating the importance of mass spectrometry as a tool for discovery.