Comprehensive yeast proteome analysis using a capillary isoelectric focusing‐based multidimensional separation platform coupled with ESI‐MS/MS

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Abstract
As demonstrated in this study, a CIEF‐based multidimensional separation platform not only is compatible with the detergent‐based membrane protein preparation protocol, but also achieves both the largest yeast membrane proteome coverage and the most comprehensive analysis of the yeast proteome to date. By using a 1% false discovery rate for total peptide identifications, a total of 2513 distinct yeast proteins are identified from the SDS‐solubilized fraction with an average of 5.4 peptides leading to each protein identification. Among proteins identified from the SDS‐solubilized fraction, 407 proteins are predicted to contain at least two or more transmembrane domains using TMHMM (www.cbs.dtu.dk/services/TMHMM‐2.0/), corresponding to 46% yeast membrane proteome coverage. Only four additional membrane proteins are identified in the soluble and urea‐solubilized fractions, affirming the utility of SDS extraction for enriching the membrane proteome. By combining proteome results obtained from the soluble, urea‐solubilized, and SDS‐solubilized fractions, a single yeast proteome analysis yields the identification of 3632 distinct yeast proteins, corresponding to 55% theoretical yeast proteome coverage or 70% of proteins predicted to be expressed during log‐phase growth in rich media.

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