Large-scale evaluation of quantitative reproducibility and proteome coverage using acid cleavable isotope coded affinity tag mass spectrometry for proteomic profiling.
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.
Molloy
Mark P
MP
Pfizer Global Research and Development, Department of Molecular Technologies, Michigan Laboratories, Ann Arbor, MI, USA. mmolloy@proteome.org.au <mmolloy@proteome.org.au>
Donohoe
Sam
S
Brzezinski
Erin E
EE
Kilby
Greg W
GW
Stevenson
Tracy I
TI
Baker
J David
JD
Goodlett
David R
DR
Gage
Douglas A
DA
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
D000891:Anti-Infective Agents, Local; D029968:Escherichia coli Proteins; D010455:Peptides; D020543:Proteome; D014260:Triclosan