A rapid, reproducible, on-the-fly orthogonal array optimization method for targeted protein quantification by LC/MS and its application for accurate and sensitive quantification of carbonyl reductases in human liver.

Liquid chromatography (LC)/mass spectrometry (MS) in selected-reactions-monitoring (SRM) mode provides a powerful tool for targeted protein quantification. However, efficient, high-throughput strategies for proper selection of signature peptides (SP) for protein quantification and accurate optimization of their SRM conditions remain elusive. Here we describe an on-the-fly, orthogonal array optimization (OAO) approach that enables rapid, comprehensive, and reproducible SRM optimization of a large number of candidate peptides in a single nanoflow-LC/MS run. With the optimized conditions, many peptide candidates can be evaluated in biological matrixes for selection of the final SP. The OAO strategy employs a systematic experimental design that strategically varies product ions, declustering energy, and collision energy in a cycle of 25 consecutive SRM trials, which accurately reveals the effects of these factors on the signal-to-noise ratio of a candidate peptide and optimizes each. As proof of concept, we developed a highly sensitive, accurate, and reproducible method for the quantification of carbonyl reductases CBR1 and CBR3 in human liver. Candidate peptides were identified by nano-LC/LTQ/Orbitrap, filtered using a stringent set of criteria, and subjected to OAO. After evaluating both sensitivity and stability of the candidates, two SP were selected for quantification of each protein. As a result of the accurate OAO of assay conditions, sensitivities of 80 and 110 amol were achieved for CBR1 and CBR3, respectively. The method was validated and used to quantify the CBRs in 33 human liver samples. The mean level of CBR1 was 93.4 +/- 49.7 (range: 26.2-241) ppm of total protein, and of CBR3 was 7.69 +/- 4.38 (range: 1.26-17.9) ppm. Key observations of this study: (i) evaluation of peptide stability in the target matrix is essential for final selection of the SP; (ii) utilization of two unique SP contributes to high reliability of target protein quantification; (iii) it is beneficial to construct calibration curves using standard proteins of verified concentrations to avoid severe biases that may result if synthesized peptides alone are used. Overall, the OAO method is versatile and adaptable to high-throughput quantification of validated biomarkers identified by proteomic discovery experiments.

Genetic polymorphisms in the carbonyl reductase 3 gene CBR3 and the NAD(P)H:quinone oxidoreductase 1 gene NQO1 in patients who developed anthracycline-related congestive heart failure after childhood cancer.

BACKGROUND: Exposure to anthracyclines as part of cancer therapy has been associated with the development of congestive heart failure (CHF). The potential role of genetic risk factors in anthracycline-related CHF remains to be defined. Thus, in this study, the authors examined whether common polymorphisms in candidate genes involved in the pharmacodynamics of anthracyclines (in particular, the nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 gene NQO1 and the carbonyl reductase 3 gene CBR3) had an impact on the risk of anthracycline-related CHF. METHODS: A nested case-control study was conducted within a cohort of 1979 patients enrolled in the Childhood Cancer Survivor Study who received treatment with anthracyclines and had available DNA. Thirty patients with CHF (cases) and 115 matched controls were genotyped for polymorphisms in NQO1 (NQO1*2) and CBR3 (the CBR3 valine [V] to methionine [M] substitution at position 244 [V244M]). Enzyme activity assays with recombinant CBR3 isoforms (CBR3 V244 and CBR3 M244) and the anthracycline substrate doxorubicin were used to investigate the functional impact of the CBR3 V244M polymorphism. RESULTS: Multivariate analyses adjusted for sex and primary disease recurrence were used to test for associations between the candidate genetic polymorphisms (NQO1*2 and CBR3 V244M) and the risk of CHF. Analyses indicated no association between the NQO1*2 polymorphism and the risk of anthracycline-related CHF (odds ratio [OR], 1.04; P=.97). There was a trend toward an association between the CBR3 V244M polymorphism and the risk of CHF (OR, 8.16; P=.056 for G/G vs A/A; OR, 5.44; P=.092 for G/A vs A/A). In line, recombinant CBR3 V244 (G allele) synthesized 2.6-fold more cardiotoxic doxorubicinol per unit of time than CBR3 M244 (A allele; CBR3 V244 [8.26+/-3.57 nmol/hour.mg] vs CBR3 M244 [3.22+/-0.67 nmol/hour.mg]; P=.01). CONCLUSIONS: The functional CBR3 V244M polymorphism may have an impact on the risk of anthracycline-related CHF among childhood cancer survivors by modulating the intracardiac formation of cardiotoxic anthracycline alcohol metabolites. Larger confirmatory case-control studies are warranted.