BACKGROUND: Mass spectrometry has become a powerful tool for the analysis of 
large numbers of proteins in complex samples, enabling much of proteomics. Due 
to various analytical challenges, so far no proteome has been sequenced 
completely. O'Shea, Weissman and co-workers have recently determined the copy 
number of yeast proteins, making this proteome an excellent model system to 
study factors affecting coverage.
RESULTS: To probe the yeast proteome in depth and determine factors currently 
preventing complete analysis, we grew yeast cells, extracted proteins and 
separated them by one-dimensional gel electrophoresis. Peptides resulting from 
trypsin digestion were analyzed by liquid chromatography mass spectrometry on a 
linear ion trap-Fourier transform mass spectrometer with very high mass accuracy 
and sequencing speed. We achieved unambiguous identification of more than 2,000 
proteins, including very low abundant ones. Effective dynamic range was limited 
to about 1,000 and effective sensitivity to about 500 femtomoles, far from the 
subfemtomole sensitivity possible with single proteins. We used SILAC (stable 
isotope labeling by amino acids in cell culture) to generate one-to-one pairs of 
true peptide signals and investigated if sensitivity, sequencing speed or 
dynamic range were limiting the analysis.
CONCLUSION: Advanced mass spectrometry methods can unambiguously identify more 
than 2,000 proteins in a single proteome. Complex mixture analysis is not 
limited by sensitivity but by a combination of dynamic range (high abundance 
peptides preventing sequencing of low abundance ones) and by effective 
sequencing speed. Substantially increased coverage of the yeast proteome appears 
feasible with further development in software and instrumentation.