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

Proteomics. 2005 Apr;5(5):1204-8. doi: 10.1002/pmic.200400994.


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.

Publication types

  • Evaluation Study

MeSH terms

  • Anti-Infective Agents, Local / pharmacology
  • Escherichia coli / drug effects
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / analysis*
  • Isotope Labeling / methods*
  • Mass Spectrometry / methods*
  • Peptides / analysis
  • Proteome / analysis*
  • Reproducibility of Results
  • Software
  • Triclosan / pharmacology


  • Anti-Infective Agents, Local
  • Escherichia coli Proteins
  • Peptides
  • Proteome
  • Triclosan