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

Abstract

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 matrices for selection of the final SP. The OAO strategy employs a systematic experimental design that strategically varies product ions, de-clustering energy and collision energy in a cycle of 25 consecutive SRM trials, which accurately reveals the effects of these factors on the single-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 for CBR3 was 7.69±4.38 (range: 1.26–17.9) ppm. Key observations of this study are that: 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; and 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.

Nano-scale liquid chromatography/mass spectrometry and on-the-fly orthogonal array optimization for quantification of therapeutic monoclonal antibodies and the application in preclinical analysis

“…A predefined L 25 (3 5 ) orthogonal array design [28] was applied to evaluate the critical SRM parameters including product ion, collision energy (CE) and tube-lens voltage. The range of CE was estimated based on the empirical values we established through pilot analyses of 103 unique tryptic peptides [24]. The optimization was conducted by programming 25 independent SRM measurements with strategically varied parameters.…”

“…Firstly, most methods use a lone SP for the quantification of a protein, which may carry a significant risk of error where proteins are truncated biologically outside the SP domain [21,22] or certain residues within the SP domain are biologically modified[23]. Secondly, the majority of current approaches use synthesized peptides as calibrators, which may lead to severe negative biases, as we observed previously [24]. Finally, due to the high molecule weight of therapeutic proteins and their relatively low concentrations in pharmaceutical samples, the sensitivity achievable by a conventional LC/MS is often insufficient, especially for these studies employing low therapeutic doses [5].…”

“…A highly sensitive nano-LC/Nanospray-MS method was employed to achieve a low lower limit of quantification (LLOQ). An on-the-fly orthogonal array optimization (OAO) method that we developed recently [24] was utilized for the high-throughput optimization of SRM conditions for each SP candidate. Without using synthesized peptides, the OAO approach is able to achieve batch-wise development of SRM methods, e.g.…”

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics

“…14,15,33 b pI values were calculated with a tool from https://web.expasy.org/compute_pi/ c Grand-average-of-hydropathy (GRAVY) values were acquired with a tool by Dr. Stephan Fuchs (http://www.gravy-calculator.de). …”

Phosphorylation of Tyr188 in the WW domain of YAP1 plays an essential role in YAP1-induced cellular transformation

“…The band containing YAP1 were excised and digested with in-gel digestion protocol with trypsin only and solution samples were divided into 2 aliquots for dual enzyme digestion of trypsin and GluC according to the 2-step on pellet digestion protocol. 22,23 The digests were eluted on a 100 cm column with a 2-hr gradient with CID and ETD activation by Fusion Tribrid mass spectrometer (Thermo Scientific), using a previously described condition. 24 Raw files were searched with Proteome Discoverer (Thermo Scientific) against YAP1 sequence and Carbamidomethyl (57.021Da) was set as static modification on Cystine (C), and oxidation (15.995Da ) on methionine (M); phosphorylation (79.966 Da) on serine (S), threonine (T) and tyrosine (Y) were set as dynamic side chain modifications.…”