Combination of Improved ¹⁸O Incorporation and Multiple Reaction Monitoring: A Universal Strategy for Absolute Quantitative Verification of Serum Candidate Biomarkers of Liver Cancer

Abstract

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS), which is an alternative to immunoassay methods such as ELISA and Western blotting, has been used to alleviate the bottlenecks of high-throughput verification of biomarker candidates recently. However, the inconvenience and high isotope consumption required to obtain stably labeled peptide impedes the broad application of this method. In our study, the (18)O-labeling method was introduced to generate stable isotope-labeled peptides instead of the Fmoc chemical synthesis and Qconcat recombinant protein synthesis methods. To make (18)O-labeling suitable for absolute quantification, we have added the following procedures: (1) RapiGest SF and microwave heating were added to increase the labeling efficiency; (2) trypsin was deactivated completely by chemical modification using tris(2-carboxyethyl)phosphine (TCEP) and iodoacetamide (IAA) to prevent back-exchange of (18)O to (16)O, and (3) MRM parameters were optimized to maximize specificity and better distinguish between (18)O-labeled and unlabeled peptides. As a result, the (18)O-labeled peptides can be prepared in less than 1 h with satisfactory efficiency (>97%) and remained stable for 1 week, compared to traditional protocols that require 5 h for labeling with poor stability. Excellent separation of (18)O-labeled and unlabeled peptides was achieved by the MRM-MS spectrum. Finally, through the combined improvement in (18)O-labeling with multiple reaction monitoring, an absolute quantification strategy was developed to quantitatively verify hepatocellular carcinoma-related biomarker candidates, namely, vitronectin and clusterin, in undepleted serum samples. Sample preparation and capillary-HPLC analysis were optimized for high-throughput applications. The reliability of this strategy was further evaluated by method validation, with accuracy (%RE) and precision (%RSD) of less than 20% and good linearity (r(2) > 0.99), and clinical validation, which were consistent with previously reported results. In summary, our strategy can promote broader application of SID-MRM-MS for biomarkers from discovery to verification regarding the significant advantages of the convenient and flexible generation of internal standards, the reduction in the sample labeling steps, and the simple transition.

Biomarker Discovery for Early Detection of Hepatocellular Carcinoma in Hepatitis C–infected Patients

“…However, antibody reagents with sufficient specificity for the target proteins or their peptides are generally not available, and the de- velopment of such reagents is expensive and time consuming. The capacity of SRM for multiplexed, high-throughput analysis, together with its sensitivity and quantification, positions SRM as a promising application in medical screening (24,25).…”

Protein Analysis by Shotgun/Bottom-up Proteomics

“…This strategy employs synthetic unlabeled peptides which may be labeled with isotope reagent, such as iTRAQ 54 or 18 O water. 515 The synthesized peptide and the endogenous peptide sample, including its counterpart, are labeled with different isotopes by chemical or enzymatic reactions. The advantage of this method is the use of unlabeled synthetized peptides and readily available isotopic labeling reagents.…”

18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

“…However, the technology is presently limited in the number of candidate proteins that can be screened often because of the costs associated with stable isotopelabeled synthetic peptides (1,3,7,12,13) or artificial protein concatemers (1,14,15) that are typically utilized as internal standards. Recently the use of 18 O labeling for individual synthetic peptides as internal standards for SRM has also been recently reported (16). In this work, we investigated the concept of using an 18 O-labeled proteome reference to generate global internal standards (GIS) as an alternative to stable isotope-labeled synthetic peptides for broad SRM-based relative quantification.…”