Mass spectrometric identification of cellular proteins: Sample preparation and data analysis techniques for proteomics.

摘要

The recent publication of the complete genomes of several organisms has facilitated an explosion in the application of mass spectrometry to the study of proteins. Qualitative proteomics seeks to characterize the composition of the protein complement of biological systems. This work explores two different types of problems in protein characterization that are amenable to mass spectrometric study. The first set of experiments elucidates the proteins present in the stalk substructure of Caulobacter crescentus bacteria. Stalk proteins were separated by two-dimensional electrophoresis prior to digestion with the enzyme trypsin. Proteins are identified by peptide mass fingerprinting, i.e. comparing observed tryptic peptide masses to those predicted from the Caulobacter genome. A novel chemical derivatization allows elucidation of the lysine content of the tryptic fragments, and the lysine content information facilitates protein identification. An exhaustive analysis of observed masses that did not initially correspond to Caulobacter stalk protein tryptic peptides illuminates many of challenges that face researchers in peptide mass fingerprinting experiments. A second set of experiments was undertaken to localize phosphorylated residues in a particular Arabidopsis protein. This work led to a study of the impact of a side reaction called deamidation on peptide mass fingerprinting. This side reaction occurs readily during isolation of phosphorylated peptides under basic conditions. A key aspect of any analytical methodology is sample preparation. The current state of the art in protein separation, two-dimensional gel electrophoresis, has several significant shortcomings. A novel two-dimensional liquid phase separation system was constructed and characterized. This apparatus used different diameter columns in the two dimensions to concentrate proteins during the separation prior to mass spectrometric analysis.