Abstract
Currently, the field of shotgun proteomics relies primarily on the separation of peptides by reversed-phase microcapillary chromatography (RP-μLC) combined with either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) and tandem mass spectrometry (MS/MS) for protein identification as well as quantification. For this purpose we herein describe construction of a RP-μLC–ESI column-emitter along with optimized μLC conditions for using the device to quantify pair-wise changes in protein expression via the isotope coded affinity tag (ICAT™) method that also maximize peak capacity. These optimized RP-μLC parameters required a balance be reached between the disparate needs of quantification which requires good peak shape and identification (i.e. proteome coverage) of proteins via peptide collision induced dissociation (CID) which requires peak capacity be maximized. A complex biological sample from a study of murine acetaminophen toxicity in hepatocyes was chosen for method development because of the high level complexity, but the biological results are not the focus of this manuscript.
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