A new sample preparation method for the absolute quantitation of a target proteome using ¹⁸O labeling combined with multiple reaction monitoring mass spectrometry

Abstract

A key step in the workflow of bottom-up proteomics is the proteolysis of proteins into peptides with trypsin. In addition, enzyme-catalytic (18)O labeled peptides as internal standards coupled with multiple reaction monitoring mass spectrometry (MRM MS) for the absolute quantitation of the target proteome is commonly used for its convenient operation and low cost. However, long digestion and labeling times, incomplete digestion and (18)O to (16)O back exchange limit its application, therefore, we developed a rapid and efficient digestion method based on a high ratio of trypsin to protein. In addition, after separation of the digested samples using pipette tips packed with reversed-phase packing materials in house, the trypsin can be separated, collected and reused at least four times. Based on this approach, a novel protein quantification method using (18)O-labeled QconCAT peptides as internal standards combined with MRM MS for the absolute quantitation of a target proteome is established. Experimental results showed that the novel method had high digestion and (18)O labeling efficiencies, and no (18)O to (16)O back-exchange occurred. A linear range covering 2 orders of magnitude and a limit of quantification (LOQ) as low as 5 fmol were achieved with an RSD below 10%. Then, the quantitative method is used for the absolute quantitation of drug metabolizing enzymes in human liver microsomes. The results are in good agreement with the previously reported data, which demonstrates that the novel method can be used for absolute quantitative analyses of target proteomes in complex biological samples.

Proteomics of endometrial cancer diagnosis, treatment, and prognosis

“…To date, biomarkers have proven tremendous clinical value in early diagnosis [3,4], categorizing different subtypes of malignancies [5,6], and in monitoring patient's response to therapy [7,8]. Regardless of the numerous proteomic studies that have contributed to the standardization of experimental protocols for digestion [9], separation [10], enrichment [11], identification [12], and quantification [13] of low abundant proteins by highly efficient mass spectrometric techniques, proteomic research is still restricted by both technology and bioinformatics tools. Many protein and peptide peaks have been reported to bear significant diagnostic [14], prognostic [15], or predictive value [16] for EC; however, the candidate biomarkers have not yet been validated for use in clinical patient care [17].…”

From Genotype to Phenotype: Content and Activities of Cytochromes P450 2A6 in Human Liver In Vitro and Predicted In Vivo

“…Protein quantification of CYP2A6 was performed by nano-liquid chromatography (LC)-mass spectrometry (MS)/MS as previously reported (Zhang et al, 2016). A QconCAT protein consisting of 57 stable isotope-labeled peptides from 21 drug-metabolizing enzymes (including CYP2A6) in which two or three peptides were selected for each targeted protein was employed to quantify these proteins in HLMs (Li et al, 2015). The concentration of CYP2A6 protein was determined by nano-LC-multiple reaction monitoring MS using an easy nano-LC (Thermo Fisher Scientific Inc., Waltham, MA) coupled to a TSQVantage Triple Quadrupole mass spectrometer (Thermo Fisher Scientific Inc.).…”

A new calibration curve calculation method for absolute quantification of drug metabolizing enzymes in human liver microsomes by stable isotope dilution mass spectrometry

“…27 The genes encoding the QconCAT protein were rst inserted into prokaryotic expression plasmids by Sangon Biotech Ltd. (Shanghai, China), and then the plasmids were transformed and expressed in E. coli in DMEM medium containing 13 C 6 L-lysine and 13 C 6 L-arginine. 27 The genes encoding the QconCAT protein were rst inserted into prokaryotic expression plasmids by Sangon Biotech Ltd. (Shanghai, China), and then the plasmids were transformed and expressed in E. coli in DMEM medium containing 13 C 6 L-lysine and 13 C 6 L-arginine.…”

“…To quantify 21 drug metabolizing enzymes in human liver microsomes, QconCAT proteins were designed as a concatemer of 57 stable isotope-labeled peptides, and two or three peptides were selected for each targeted protein. 27 The genes encoding the QconCAT protein were rst inserted into prokaryotic expression plasmids by Sangon Biotech Ltd. (Shanghai, China), and then the plasmids were transformed and expressed in E. coli in DMEM medium containing 13 C 6 L-lysine and 13 C 6 L-arginine. The QconCAT protein was puried using affinity chromatography and conrmed using matrix-assisted laser desorption ionization-time of ight mass spectrometry (MALDI-TOF MS) similarly to methods described in the literature.…”