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Hepatocellular carcinoma (HCC) is a malignant cancer with one of the highest mortality rates. Des‐γ‐carboxyprothrombin (DCP) is an HCC serologic surveillance marker that can complement the low sensitivity of alpha‐fetoprotein (AFP). DCP exists in the blood as a mixture of proteoforms from an impaired carboxylation process at glutamic acid (Glu) residues within the N‐terminal domain. The heterogeneity of DCP may affect the accuracy of measurements because DCP levels are commonly determined using an immunoassay that relies on antibody reactivity to an epitope in the DCP molecule. In this study, we aimed to improve the DCP measurement assay by applying a mass spectrometry (MS)‐based approach for a more inclusive quantification of various DCP proteoforms. We developed a multiple‐reaction monitoring–MS (MRM‐MS) assay to quantify multiple noncarboxylated peptides included in the various des‐carboxylation states of DCP. We performed the MRM‐MS assay in 300 patients and constructed a robust diagnostic model that simultaneously monitored three noncarboxylated peptides. The MS‐based quantitative assay for DCP had reliable surveillance power, which was evident from the area under the receiver operating characteristic curve (AUROC) values of 0.874 and 0.844 for the training and test sets, respectively. It was equivalent to conventional antibody‐based quantification, which had AUROC values at the optimal cutoff (40 mAU/mL) of 0.743 and 0.704 for the training and test sets, respectively. The surveillance performance of the MS‐based DCP assay was validated using an independent validation set consisting of 318 patients from an external cohort, resulting in an AUROC value of 0.793. Conclusion: Due to cost effectiveness and high reproducibility, the quantitative DCP assay using the MRM‐MS method is superior to antibody‐based quantification and has equivalent performance.

There is an urgent need for new biomarkers that address the shortcomings of current screening methods which fail to detect a large proportion of cases with hepatocellular carcinoma (HCC) at early stage. To develop a robust, multiple‐biomarker panel based on multiple reaction monitoring–mass spectrometry with high performance in detecting early‐stage HCC within at‐risk populations. In the discovery set, 150 samples were analyzed to identify candidate biomarkers. The resulting list of candidates was tested in the training set (713 samples) to establish a multimarker panel, which was evaluated in the validation set (305 samples). We identified 385 serum HCC biomarker candidates in the discovery set and developed a multimarker panel consisting of 28 peptides that best differentiated HCC from controls. The area under the receiver operating characteristic curve of multimarker panel was significantly higher than alpha‐fetoprotein (AFP) in the training (0.976 vs. 0.804; P