HST-MRM-MS: A Novel High-Sample-Throughput Multiple Reaction Monitoring Mass Spectrometric Method for Multiplex Absolute Quantitation of Hepatocellular Carcinoma Serum Biomarker.

Absolute quantification of clinical biomarkers by mass spectrometry (MS) has been challenged due to low sample-throughput of current multiple reaction monitoring (MRM) methods. For this problem to be overcome, in this work, a novel high-sample-throughput multiple reaction monitoring mass spectrometric (HST-MRM-MS) quantification approach is developed to achieve simultaneous quantification of 24 samples. Briefly, triplex dimethyl reagents (L, M, and H) and eight-plex iTRAQ reagents were used to label the N- and C-termini of the Lys C-digested peptides, respectively. The triplex dimethyl labeling produces three coelute peaks in MRM traces, and the iTRAQ labeling produces eight peaks in MS2, resulting in 24 (3×8) channels in a single experiment. HST-MRM-MS has shown good accuracy ( R2 > 0.98 for absolute quantification), reproducibility (RSD < 15%), and linearity (2-3 orders of magnitude). Moreover, the novel method has been successfully applied in quantifying serum biomarkers in hepatocellular carcinoma (HCC)-related serum samples. In conclusion, HST-MRM-MS is an accurate, high-sample-throughput, and broadly applicable MS-based absolute quantification method.