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

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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.