Increased quantitative proteome coverage with13C/12C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme

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Abstract
Quantitative protein profiling using the isotope‐coded affinity tag (ICAT™) method and tandem mass spectrometry (MS) enables the pair‐wise comparison of protein expression levels in biological samples. A new version of the ICAT reagent with an acid‐cleavable bond, which allows removal of the biotin moiety prior to MS and which utilizes 13C substitution for 12C in the heavy‐ICAT reagent rather than 2H (for 1H) as in the original reagent (Gygi, S. P., Rist, B., Gerber, S. A., Frantisek, T., Gelb, M. H., Aebersold, R., Quantitative analysis of complex protein mixtures using isotope‐coded affinity tags. Nat. Biotechnol. 1999, 17, 994–999), was investigated. We developed and validated an MS data acquisition strategy using this new reagent that results in an increased number of protein identifications per experiment, without losing the accuracy of protein quantification. This was achieved by following a single survey (precursor) ion scan and serial collisioninduced dissociations (CIDs) of four different precursor ions observed in the prior survey scan. This strategy is common to many high‐performance liquid chromatography‐electrospray ionization (HPLC‐ESI)‐MS shotgun proteomic strategies, but heretofore not to ICAT experiments. This advance is possible because the new ICAT reagent uses 13C as the “heavy” element rather than 2H, thus, eliminating the slight delay in retention time of ICAT‐labeled “light” peptides on a C18‐based HPLC separation that occurs with 2H and 1H. Analyses using this new scheme of an ICAT‐labeled trypsin‐digested six protein mixture as well as a tryptic digest of a total yeast lysate, indicated that about two times more proteins were identified in a single analysis, and that there was no loss in accuracy of quantification.