Development of mass spectrometry-based proteomics and its applications in biological systems.

摘要

In the past decade, there have been remarkable advances in mass spectrometry-based proteomic technologies. The complexity of most proteomes, however, still far exceeds the capability of current technologies to fully resolve the individual constituents. Therefore, further improvements in proteomic platforms are necessary to achieve in-depth coverage of the whole proteome. To accomplish this, we coupled ion mobility spectrometry (IMS) to multidimensional liquid chromatography--mass spectrometry (LC-MS/MS). A salient feature with IMS inclusion is increased peak capacity in the separation scheme. Such an approach was evaluated by analyzing the plasma proteome. We observed substantially improved proteome coverage by detecting ∼9000 plasma proteins, of which ∼3000 were established as high-confidence assignments. Clearly, our IMS approach holds promise for comprehensive proteome analysis.;By no means is proteomics just providing inventories of proteins in biological systems. Their expression levels are of considerable interest to understanding the molecular basis of biological processes. We studied protein expression associated with a mouse model of Huntington's disease (HD) by quantitative MS. Protein levels in both brain tissue and plasma were examined with LC-MS/MS-based approaches. Intriguingly, we found that in contrast to the altered proteins in the brain, proteomic alterations in the plasma were less relevant to HD pathology. Rather, they were more likely due to secondary effects of the disease (e.g., inflammation), thereby reflecting a more systemic view of HD.;Efforts have also been devoted to studying membrane proteins as they are often underrepresented in proteomic analysis. Even after enrichment, cytosolic contaminants still comprise a large fraction (∼40%) of the sample. We chose to work on pathogenic Chlamydia trachomatis due to its distinct membrane structure. Unlike typical cytoplasmic membranes, the chlamydial outer membrane is highly cross-linked and affords extra stability and rigidity, thereby allowing efficient preparation of the membrane fraction by selectively solubilizing the cytoplasm. LC-MS/MS analysis showed that >97% of resulting protein assignments were indeed due to membrane components (3% contaminants). The novel membrane proteins we found should greatly aid in future vaccine development.