title: Large-scale evaluation of quantitative reproducibility and proteome coverage using acid cleavable isotope coded affinity tag mass spectrometry for proteomic profiling.
abstract: Strategies employing non-gel based methods for quantitative proteomic profiling such as isotope coded affinity tags coupled with mass spectrometry (ICAT-MS) are gaining attention as alternatives to two-dimensional gel electrophoresis (2-DE). We have conducted a large-scale investigation to determine the degree of reproducibility and depth of proteome coverage of a typical ICAT-MS experiment by measuring protein changes in Escherichia coli treated with triclosan, an inhibitor of fatty acid biosynthesis. The entire ICAT-MS experiment was conducted on four independent occasions where more than 24 000 peptides were quantitated using an ion-trap mass spectrometer. Our results demonstrated that quantitatively, the technique provided good reproducibility (median coefficient of variation of ratios was 18.6%), and on average identified more than 450 unique proteins per experiment. However, the method was strongly biased to detect acidic proteins (pI < 7), under-represented small proteins (<10 kDa) and failed to show clear superiority over 2-DE methods in monitoring hydrophobic proteins from cell lysates.
authors.affiliation: Pfizer Global Research and Development, Department of Molecular Technologies, Michigan Laboratories, Ann Arbor, MI, USA. mmolloy@proteome.org.au <mmolloy@proteome.org.au>
authors.forename: Mark P
authors.initials: MP
authors.lastname: Molloy
authors.forename: Sam
authors.initials: S
authors.lastname: Donohoe
authors.forename: Erin E
authors.initials: EE
authors.lastname: Brzezinski
authors.forename: Greg W
authors.initials: GW
authors.lastname: Kilby
authors.forename: Tracy I
authors.initials: TI
authors.lastname: Stevenson
authors.forename: J David
authors.initials: JD
authors.lastname: Baker
authors.forename: David R
authors.initials: DR
authors.lastname: Goodlett
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Gage
mesh_terms: D000891:Anti-Infective Agents, Local / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D012984:Software; D014260:Triclosan / Q000494:pharmacology
chemical_list: D000891:Anti-Infective Agents, Local; D029968:Escherichia coli Proteins; D010455:Peptides; D020543:Proteome; D014260:Triclosan