Comprehensive yeast proteome analysis using a capillary isoelectric focusing-based multidimensional separation platform coupled with ESI-MS/MS.
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.
Wang
Weijie
W
Calibrant Biosystems, Gaithersburg, MD 20878, USA.
Guo
Tong
T
Song
Tao
T
Lee
Cheng S
CS
Balgley
Brian M
BM
D000595:Amino Acid Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000235:genetics; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010455:Peptides; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins