10.1021/ac902314m.s001
Jin Cao
Jin
Cao
Vanessa M. Covarrubias
Vanessa M.
Covarrubias
Robert M. Straubinger
Robert M.
Straubinger
Hao Wang
Hao
Wang
Xiaotao Duan
Xiaotao
Duan
Haoying Yu
Haoying
Yu
Jun Qu
Jun
Qu
Javier G. Blanco
Javier G.
Blanco
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
American Chemical Society
2010
OAO
Targeted Protein Quantification
candidate
target protein quantification
peptide
proteomic discovery experiments
orthogonal array optimization
method
carbonyl reductases CBR 1
Human LiverLiquid chromatography
MS
SRM
CBR 3
CBR 1
SP
protein quantification
LC
2010-04-01 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/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/2780710
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.