Pharmaceutical Sciences Faculty Publications
Evaluation of the Acid-Cleavable Isotope-Coded Affinity Tag Reagents: Application to Camptothecin-Treated Cortical Neurons
Document Type
Article
Publication Date
5-1-2004
Journal Title
Journal of Proteome Research
ISSN
1535-3893
Volume
3
Issue
3
First Page
469
Last Page
477
DOI
10.1021/pr034090t
PubMed ID
15253428
Abstract
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 muRPLC-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 muRPLC-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
Amino acid sequence, camptothecin, carbon isotopes, gene expression regulation, isotopes, neurons, proteome, serum albumin, bovine, spectrometry, mass
Recommended Citation
Yu, Li-Rong; Conrads, Thomas P.; Uo, Takuma; Issaq, Haleem J.; Morrison, Richard S.; and Veenstra, Timothy D., "Evaluation of the Acid-Cleavable Isotope-Coded Affinity Tag Reagents: Application to Camptothecin-Treated Cortical Neurons" (2004). Pharmaceutical Sciences Faculty Publications. 468.
https://digitalcommons.cedarville.edu/pharmaceutical_sciences_publications/468