Journal Article

Relative and absolute quantitative shotgun proteomics: targeting low-abundance proteins in <i>Arabidopsis thaliana</i>

Stefanie Wienkoop and Wolfram Weckwerth

in Journal of Experimental Botany

Published on behalf of Society for Experimental Biology

Volume 57, issue 7, pages 1529-1535
Published in print April 2006 | ISSN: 0022-0957
Published online March 2006 | e-ISSN: 1460-2431 | DOI: http://dx.doi.org/10.1093/jxb/erj157
Relative and absolute quantitative shotgun proteomics: targeting low-abundance proteins in Arabidopsis thaliana

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The plant system is a highly dynamic structure on all molecular levels, transcripts, proteins, and metabolites. Thus, protein analysis has to cope with a highly dynamic range of concentrations. A severe problem is the detection of low-abundance proteins in the presence of housekeeping proteins. Basically three approaches are facilitated to measure protein abundance in a comprehensive manner: 2DE and one- or multi-dimensional shotgun proteomics, with or without stable-isotope labelling. These comparative techniques allow for the identification of altered protein levels compared with a reference state. However, they are limited to the analysis of medium/high-abundance proteins. Using stable-isotope dilution techniques it is possible to target the quantitative analysis to low-abundance proteins and to measure absolute concentrations of proteins. Based on multi-dimensional non-gel shotgun proteomics in Arabidopis thaliana, a list of tryptic peptides comprising >1000 proteins was generated. A strategy for quantitative plant proteomics is proposed using this master-list for selecting signature peptides of proteins. To prove the concept, a liquid chromatography–high-resolution triple quadrupole multiple reaction monitoring–mass spectrometry technique is described to determine the absolute amount of a low-abundance sucrose synthase isoform out of an ultra-complex A. thaliana protein extract.

Keywords: Absolute quantitation; Arabidopsis; high resolution; linear ion trap; multiple reaction monitoring (MRM); plant; quantitative proteomics; relative quantitation; shotgun proteomics; single reaction monitoring (SRM); stable-isotope labelling; SUSY; triple quadrupole

Journal Article.  3678 words.  Illustrated.

Subjects: Plant Sciences and Forestry

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