Yeast remains an important model for systems biology and for evaluating 
proteomics strategies. In-depth shotgun proteomics studies have reached nearly 
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., 
Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., and Mann, M. 
(2011) Deep and highly sensitive proteome coverage by LC-MS/MS without 
prefractionation. Mol. Cell Proteomics 10, 10.1074/mcp.M110.003699). Here we 
couple an ultra high pressure liquid chromatography system to a novel bench top 
Orbitrap mass spectrometer (Q Exactive) with the goal of nearly complete, rapid, 
and robust analysis of the yeast proteome. Single runs of filter-aided sample 
preparation (FASP)-prepared and LysC-digested yeast cell lysates identified an 
average of 3923 proteins. Combined analysis of six single runs improved these 
values to more than 4000 identified proteins/run, close to the total number of 
proteins expressed under standard conditions, with median sequence coverage of 
23%. Because of 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 Kyoto Encyclopedia of Genes and Genomes 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 widespread use in the yeast and other 
biological research communities.