Comprehensive proteornics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing

被引:69
作者
Breci, L
Hattrup, E
Keeler, M
Letarte, J
Johnson, R
Haynes, PA
机构
[1] Univ Arizona, Dept Biochem & Mol Biophys, Tucson, AZ 85721 USA
[2] Univ Arizona, Dept Chem, Tucson, AZ 85721 USA
[3] Univ Arizona, Bio5 Inst Collaborat Biores, Tucson, AZ 85721 USA
关键词
gas phase fractionation; protein identification; tandem mass spectrometry; two-dimensional chromatography;
D O I
10.1002/pmic.200401103
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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; 390proteins 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.
引用
收藏
页码:2018 / 2028
页数:11
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