Large-scale evaluation of quantitative reproducibility and proteome coverage using acid cleavable isotope coded affinity tag mass spectrometry for proteomic profiling

Mark P. Molloy*, Sam Donohoe, Erin E. Brzezinski, Greg W. Kilby, Tracy I. Stevenson, J. David Baker, David R. Goodlett, Douglas A. Gage

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

55 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1204-1208
Number of pages5
JournalProteomics
Volume5
Issue number5
DOIs
Publication statusPublished - Apr 2005

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