System-wide Perturbation Analysis with Nearly Complete Coverage of the Yeast Proteome by Single-shot Ultra HPLC Runs on a Bench Top Orbitrap
Mol Cell Proteomics. 2011 Oct 20. [Epub ahead of print]
Nagaraj N, Kulak NA, Cox J, Neuhaus N, Mayr K, Hoerning O, Vorm O, Mann M.
Yeast remains an important model for systems biology and for evaluating proteomics strategies. In-depth shotgun proteomics studies have reached near-comprehensive coverage and rapid, targeted approaches have been developed for this organism. Recently we demonstrated that single LC MS/MS analysis using long columns and gradients coupled to a linear ion trap Orbitrap instrument had an unexpectedly large dynamic range of protein identification (Thakur, S.S. MCP 2011, PMID: 21586754). Here we couple an ultra high pressure liquid chromatography system to a novel bench-top Orbitrap mass spectrometer (Q Exactive) with the goal of near-complete, rapid and robust analysis of the yeast proteome. Single runs of FASP-prepared and LysC digested yeast cell lysates identified an average of 3,923 proteins. Combined analysis of six single runs improved these values to more than 4,000 identified proteins per run, close to the total number of proteins expressed under standard conditions, with median sequence coverage of 23%. Due to the absence of fractionation steps only minuscule amounts of sample are required. Thus the yeast model proteome can now largely be covered within a few hours of measurement time and at high sensitivity. Median coverage of proteins in KEGG pathways with at least 10 members was 88% and pathways not covered were not expected to be active under the conditions used. To study perturbations of the yeast proteome we developed an external, heavy-lysine labeled SILAC yeast standard representing different proteome states. This spike-in standard was employed to measure the heat shock response of the yeast proteome. Bioinformatic analysis of the heat shock response revealed that translation related functions were down-regulated, prominently including nucleolar processes. Conversely, stress related pathways were up-regulated. The proteomic technology described here is straightforward, rapid and robust, potentially enabling wide-spread use in the yeast and other biological communities.