Relative and absolute quantitative shotgun proteomics: targeting low-abundance proteins in Arabidopsis thaliana

J Exp Bot. 2006;57(7):1529-35. doi: 10.1093/jxb/erj157. Epub 2006 Mar 30.

Abstract

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.

Publication types

  • Validation Study

MeSH terms

  • Arabidopsis / chemistry
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / analysis*
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / isolation & purification*
  • Gas Chromatography-Mass Spectrometry / methods
  • Glucosyltransferases / chemistry
  • Glucosyltransferases / isolation & purification
  • Glucosyltransferases / metabolism
  • Peptides / chemistry
  • Peptides / isolation & purification
  • Peptides / metabolism
  • Proteomics / methods*
  • Sequence Analysis, Protein

Substances

  • Arabidopsis Proteins
  • Peptides
  • Glucosyltransferases
  • sucrose synthase