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Genome Biology

, 7:R50

First Online: 19 June 2006Received: 02 December 2005Revised: 21 April 2006Accepted: 19 May 2006


BackgroundMass 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.

ResultsTo 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.

ConclusionAdvanced 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.

Electronic supplementary materialThe online version of this article doi:10.1186-gb-2006-7-6-r50 contains supplementary material, which is available to authorized users.

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Autor: Lyris MF de Godoy - Jesper V Olsen - Gustavo A de Souza - Guoqing Li - Peter Mortensen - Matthias Mann


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