System-wide Perturbation Analysis with Nearly Complete Coverage of the Yeast Proteome by Single-shot Ultra HPLC Runs on a Bench Top Orbitrap *

摘要: Yeast remains an important model for systems biology and evaluating proteomics strategies. In-depth shotgun studies have reached nearly comprehensive coverage, rapid, targeted approaches been developed this organism. Recently, we demonstrated that single LC-MS/MS analysis using long columns gradients coupled to a linear ion trap Orbitrap instrument had unexpectedly large dynamic range of protein identification (Thakur, S. S., Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., Mann, M. (2011) Deep highly sensitive proteome coverage by without prefractionation. Mol. Cell Proteomics 10, 10.1074/mcp.M110.003699). Here couple ultra high pressure liquid chromatography system novel bench top mass spectrometer (Q Exactive) with the goal complete, robust yeast proteome. Single runs filter-aided sample preparation (FASP)-prepared LysC-digested cell lysates identified average 3923 proteins. Combined six improved these values more than 4000 proteins/run, close total number proteins expressed under standard conditions, median sequence 23%. Because absence fractionation steps, only minuscule amounts are required. Thus can now largely be covered within few hours measurement time at sensitivity. Median in Kyoto Encyclopedia Genes Genomes pathways least 10 members was 88%, not were expected active conditions used. To study perturbations proteome, external, heavy lysine-labeled SILAC representing different states. This spike-in employed measure heat shock response Bioinformatic revealed translation-related functions down-regulated prominently, including nucleolar processes. Conversely, stress-related up-regulated. The proteomic technology described here is straightforward, robust, potentially enabling widespread use other biological research communities.