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.
Keywords: clinical biomarker; dimethyl labeling; isobaric tags for relative and absolute quantification; multiple reaction monitoring; quantification.