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Comprehensive proteomics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing
Author(s) -
Breci Linda,
Hattrup Emily,
Keeler Matthew,
Letarte Jessica,
Johnson Roxanne,
Haynes Paul A.
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401103
Subject(s) - chromatography , chemistry , isoelectric focusing , mass spectrometry , fractionation , gel electrophoresis , polyacrylamide gel electrophoresis , two dimensional gel electrophoresis , proteome , lysis , tandem mass spectrometry , isoelectric point , proteomics , biochemistry , enzyme , gene
We have investigated the use of a variety of different techniques to identify as many proteins as possible in a yeast lysate, with the aim of investigating the overlap and complementarity of data from different approaches. A standard lysate was prepared from log phase yeast ( Saccharomyces cerevisiae ). This was then subjected to analysis via five different approaches aimed at identifying as many proteins as possible using an ion trap mass spectrometer. The total number of non‐redundant protein identifications from each experiment was: 524 proteins by 2‐D (SCX/C18) nanoflow liquid chromatography‐liquid chromatography tandem mass spectrometry (nanoLC‐LC MS/MS (MudPIT)); 381 proteins by nanoLC‐MS/MS with gas phase fractionation by mass range selection; 390 proteins by nanoLC‐MS/MS with gas phase fractionation by ion abundance selection; 898 proteins by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) separation of proteins, in‐gel digestion, and nanoLC‐MS/MS of gel slices; and 422 proteins by isoelectric focusing of proteins, in‐gel digestion and nanoLC‐MS/MS of gel slices. The total number of non‐redundant protein identifications in the five experiments was 1204. Combining only the two best experiments, the SDS‐PAGE gel slices and the Mudpit, produces 1024 proteins identified, more than 85% of the total. Clearly, combining a Mudpit analysis with an SDS‐PAGE gel slice experiment gives the greatest amount of protein identification information from a limited amount of sample.