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Evaluation of the Acid-Cleavable Isotope-Coded Affinity Tag Reagents: Application to Camptothecin-Treated Cortical Neurons

2004; American Chemical Society; Volume: 3; Issue: 3 Linguagem: Inglês

10.1021/pr034090t

1535-3907

Li‐Rong Yu, Thomas P. Conrads, Takuma Uo, Haleem J. Issaq, Richard S. Morrison, Timothy D. Veenstra,

Neuroscience and Neuropharmacology Research

The new generation of isotope-coded affinity tag (ICAT) reagents have been evaluated by labeling an equimolar amount of bovine serum albumin (BSA) with ICAT-12C9 and ICAT-13C9, combining the mixtures, digesting them with trypsin and analyzing the digestate both by μRPLC-tandem MS and by matrix-assisted laser desorption ionization (MALDI) TOF/TOF MS. The use of 13C in place of 2H resulted in both of the labeled peptides having identical elution characteristics in a reversed-phase separation. This similarity in elution allows ICAT-labeled peptides to be effectively analyzed using a μRPLC−MALDI−MS strategy as well. All of the cysteinyl-containing tryptic peptides from BSA were identified with only a 10% variation in the relative abundance measurements between the light and heavy versions of each peptide. A facile method for the removal of contaminants that arise from the cleaved biotin moiety that otherwise interfere with downstream separations and MS analysis has also been developed. The new ICAT reagents were then applied to the analysis of a cortical neuron proteome sample to identify proteins regulated by the antitumor drug, camptothecin. Keywords: isotope-coded affinity tags • quantitative proteomics • mass spectrometry • cortical neurons