期刊
JOURNAL OF EXPERIMENTAL BOTANY
卷 57, 期 7, 页码 1529-1535出版社
OXFORD UNIV PRESS
DOI: 10.1093/jxb/erj157
关键词
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
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.
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