title: Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags.
abstract: The effectiveness of proteome-wide protein identification and quantitative expression profiling is dependent on the ability of the analytical methodologies employed to routinely obtain information on low-abundance proteins, as these are frequently of great biological importance. Two-dimensional gel electrophoresis, the traditional method for proteome analysis, has proven to be biased toward highly expressed proteins. Recently, two-dimensional chromatography of the complex peptide mixtures generated by the digestion of unseparated protein samples has been introduced for the identification of their components, and isotope-coded affinity tags (ICAT) have been introduced to allow for accurate quantification of the components of protein mixtures by mass spectrometry. Here, we demonstrate that the combination of isotope coded affinity protein tags and multidimensional chromatography/mass spectrometry of tryptic peptide mixtures is capable of detecting and quantifying proteins of low abundance in complex samples.
authors.affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA. steven_gygi@hms.harvard.edu
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.forename: Beate
authors.initials: B
authors.lastname: Rist
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Griffin
authors.forename: Jimmy
authors.initials: J
authors.lastname: Eng
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis; D007554:Isotopes / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry; D015335:Molecular Probes / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D015003:Yeasts / Q000737:chemistry / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D007554:Isotopes; D015335:Molecular Probes; D020543:Proteome; D007768:Lactalbumin