Quantitative analysis of a proteome by N-terminal stable-isotope labelling of tryptic peptides.
Covalent modification of peptides and proteins with compounds containing stable isotopes (isotope tagging) has become an essential tool to detect dynamic changes in the proteome following external or internal influence; however, using terminal amino groups for global isotope labelling of tryptic peptides is challenged by the similar reactivity of the amino groups of lysine residues. We describe a new quantitative method based on selective tagging of the terminal amino groups of tryptic peptides with pentafluorophenyl esters containing stable isotopes. The labelled peptides were resolved by two-dimensional nanoflow liquid chromatography on weak anion-exchange and reversed-phase columns and then identified and quantified by tandem mass spectrometry. The method was applied to compare the proteomes of plasma membranes from proliferating and differentiated human colorectal adenocarcinoma (Caco-2) cells and endosomes purified from the livers of rats stimulated with insulin and epidermal growth factor. The comparison of the results obtained by isotope tagging and biochemical assays demonstrate that global isotope tagging with pentafluorophenyl esters allows accurate quantification of complex protein samples.
Sainte-Justine Hospital Research Center, University of Montreal, 3175 Côte Ste-Catherine Road, Montreal (Quebec), H3T 1C5, Canada.
Michael
M
Fedjaev
Stephanie
S
Trudel
Natascha
N
Tjon-A-Pan
Amanda
A
Parmar
Barry I
BI
Posner
Emile
E
Levy
Ilya
I
Nifant'ev
Alexey V
AV
Pshezhetsky
D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis*; D018938:Caco-2 Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008099:Liver / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000032:analysis*; D010455:Peptides / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry*
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D010455:Peptides; D020543:Proteome; D014357:Trypsin