Trace labeling of proteins with stable isotopes to identify fragments in complex mixtures.
We describe a novel nonradioactive protein-labeling technique that permits mass spectrometric identification of fragments of labeled proteins. Proteins are labeled by modulating their content of carbon-13 and labeled fragments identified from the distinctive isotope pattern observed on MALDI-TOF mass spectrometry. We show that carbon-13 enrichment to just 2.3% of total carbon (about twice the natural abundance of 1.1%) is sufficient for all fragments to be distinguishable from fragments of natural carbon-13-content proteins. Distinguishing labeled fragments is easily accomplished by visual inspection of spectra, but importantly, we show that labeled fragments can also be identified by computer analysis of spectra using novel parameters we have derived. The technique is demonstrated for identification of fragments of carbon-13-enriched glutathione transferase within a complex mixture of unlabeled peptides by visual and computer analysis of MALDI-TOF mass spectra, but it could be developed to mass spectrometrically identify and characterize fragments of labeled proteins recovered from biological systems.
MRC Centre for Inflammation Research, Room 231, Hugh Robson Building, University of Edinburgh, 14 George Square, Edinburgh, EH8 9JZ UK.
Juan
J
Zou
A Neil
AN
Turner
Richard G
RG
Phelps
D001331:Automation; D002247:Carbon Isotopes; D007477:Ions; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D002247:Carbon Isotopes; D007477:Ions; D010455:Peptides; D011506:Proteins; D014357:Trypsin