Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry.
The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.
Zhou
Huilin
H
Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103-8904, USA.
Ranish
Jeffrey A
JA
Watts
Julian D
JD
Aebersold
Ruedi
R
D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors
D007554:Isotopes; D010455:Peptides; D011506:Proteins; D003545:Cysteine