Relative and absolute quantitative shotgun proteomics: targeting low-abundance proteins in Arabidopsis thaliana.
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.
Proteome Factory AG, Dorotheenstr. 94, D-10117 Berlin, Germany.
Stefanie
S
Wienkoop
Wolfram
W
Weckwerth
D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein
D029681:Arabidopsis Proteins; D010455:Peptides; D005964:Glucosyltransferases; C022699:sucrose synthase