Combination of improved (18)O incorporation and multiple reaction monitoring: a universal strategy for absolute quantitative verification of serum candidate biomarkers of liver cancer.

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.