No abstract

The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ's attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.

The levels of drug-metabolizing enzymes (DMEs) and transporter proteins in the human intestine are pertinent to determine oral drug bioavailability. Despite the paucity of reports on such measurements, it is well recognized that these values are essential for translating in vitro data on drug metabolism and transport to predict drug disposition in gut wall. In the current study, clinically relevant DMEs [cytochrome P450 (P450) and uridine 59-diphospho-glucuronosyltransferase (UGT)] and drug transporters were quantified in total mucosal protein preparations from the human jejunum (n 5 4) and ileum (n 5 12) using quantification concatemer-based targeted proteomics. In contrast to previous reports, UGT2B15 and organic anion-transporting polypeptide 1 (OATP1A2) were quantifiable in all our samples. Overall, no significant disparities in protein expression were observed between jejunum and ileum. Relative mRNA expression for drug transporters did not correlate with the abundance of their cognate protein, except for P-glycoprotein 1 (P-gp) and organic solute transporter subunit alpha (OST-a), highlighting the limitations of RNA as a surrogate for protein expression in dynamic tissues with high turnover. Intercorrelations were found within P450 [2C9-2C19 (P 5 0.002, R 2 5 0.63), 2C9-2J2 (P 5 0.004, R 2 5 0.40), 2D6-2J2 (P 5 0.002, R 2 5 0.50)] and UGT [1A1-2B7 (P 5 0.02, R 2 5 0.87)] family of enzymes. There were also correlations between P-gp and several other proteins [OST-a (P < 0.0001, R 2 5 0.77), UGT1A6 (P 5 0.009, R 2 5 0.38), and CYP3A4 (P 5 0.007, R 2 5 0.30)]. Incorporating such correlations into building virtual populations is crucial for obtaining plausible characteristics of simulated individuals. SIGNIFICANCE STATEMENTA number of drug transporters were quantified for the first time in this study. Several intercorrelations of protein abundance were reported. mRNA expression levels proved to be a poor reflection of differences between individuals regarding the level of protein expression in gut. The reported abundance of drug-metabolizing enzymes and transporters and their intercorrelations will contribute to better predictions of oral drug bioavailability and drug-drug interactions by linking in vitro observations to potential outcomes through physiologically based pharmacokinetic models.