Combination of Improved 18O 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 18O-labeling method was introduced to generate stable isotope-labeled peptides instead of the Fmoc chemical synthesis and Qconcat recombinant protein synthesis methods. To make 18O-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 18O to 16O, and (3) MRM parameters were optimized to maximize specif...