RT Journal Article SR Electronic T1 Data Generated by Quantitative LC-MS Proteomics Are Only the Start and Not the Endpoint: Optimization of QconCAT-Based Measurement of Hepatic UDP-Glucuronosyltransferase Enzymes with Reference to Catalytic Activity JF Drug Metabolism and Disposition JO Drug Metab Dispos FD American Society for Pharmacology and Experimental Therapeutics SP dmd.117.079475 DO 10.1124/dmd.117.079475 A1 Brahim Achour A1 Alyssa Dantonio A1 Mark Niosi A1 Jonathan J Novak A1 Zubida M Al-Majdoub A1 Theunis C Goosen A1 Amin Rostami-Hodjegan A1 Jill Barber YR 2018 UL http://dmd.aspetjournals.org/content/early/2018/03/26/dmd.117.079475.1.abstract AB Quantitative proteomic methods require optimization at several stages, including sample preparation, LC-MS/MS and data analysis, with the final analysis stage being less widely appreciated by end-users. Achour et al. (2017b) previously reported measurement of eight uridine-5'-diphospho-glucuronosyltransferases (UGT) generated by two laboratories [using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT)], which reflected significant disparity between proteomic methods. Initial analysis of QconCAT data showed lack of correlation with catalytic activity for several UGTs (1A4, 1A6, 1A9, 2B15) and moderate correlations for UGTs 1A1, 1A3 and 2B7 (Rs=0.40-0.79, p<0.05; R2=0.30); good correlations were demonstrated between cytochrome P450 activities and abundances measured in the same experiments. Consequently, a systematic review of data analysis, starting from unprocessed LC-MS/MS data, was undertaken, with the aim of improving accuracy, defined by correlation against activity. Three main criteria were found to be important: choice of monitored peptides and fragments, correction for isotope-label incorporation, and abundance normalization using fractional protein mass. Upon optimization, abundance-activity correlations improved significantly for six UGTs (Rs=0.53-0.87, p<0.01; R2=0.48-0.73); UGT1A9 showed moderate correlation (Rs=0.47, p=0.02; R2=0.34). No spurious abundance-activity relationships were identified. However, methods remained sub-optimal for UGT1A3 and UGT1A9; here hydrophobicity of standard peptides is believed to be limiting. This commentary provides a detailed data analysis strategy and indicates, using examples, the significance of systematic data processing following acquisition. The proposed strategy offers significant improvement on existing guidelines applicable to clinically-relevant proteins quantified using QconCAT