Table of Contents
International Journal of Proteomics
Volume 2014, Article ID 451510, 11 pages
http://dx.doi.org/10.1155/2014/451510
Research Article

Comparison of Heavy Labeled (SIL) Peptide versus SILAC Protein Internal Standards for LC-MS/MS Quantification of Hepatic Drug Transporters

Department of Pharmaceutics, University of Washington, P.O. Box 357610, Seattle, WA 98195, USA

Received 13 October 2013; Revised 23 December 2013; Accepted 10 January 2014; Published 25 February 2014

Academic Editor: Andrew J. Link

Copyright © 2014 Bhagwat Prasad and Jashvant D. Unadkat. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

We studied the precision of quantification of organic anion-transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, and P-glycoprotein (P-gp) in human livers by surrogate peptide based LC-MS/MS approach using two different internal standards: stable isotope labeled peptide (SIL) versus stable isotope labeled protein (SILAC). The SIL peptides were procured commercially and the SILAC proteins were generated in-house by labeling arginine and/or lysine residues in cells expressing these transporters. Liver tissue was homogenized and the membrane fraction was isolated. The membranes were trypsin digested and the peptides were analyzed using LC-MS/MS under optimized conditions. The precision in the quantification of proteins in three independently trypsin digested samples from each liver was calculated as the standard deviation of the log transformed protein concentration. The precision of the SIL internal standard method was either slightly ( , paired t-test) better than that of the SILAC method (OATP1B1, OATP1B3, and P-gp) or not different (OATP2B1). Trypsin digestion, as measured by the response of the labeled peptide derived from the SILAC protein, was consistent across liver samples. These results indicate that when maximum trypsin digestion is ensured, the SIL internal standard method can be used with confidence for quantification of drug transporters.