Unveiling Endogenous Serum Peptides as Potential Biomarkers for Hepatocellular Carcinoma in Patients with Liver Cirrhosis

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, mainly associated with liver cirrhosis. Current diagnostic methods for HCC have limited sensitivity and specificity, highlighting the need for improved early detection and intervention. In this study, we used a comprehensive approach involving endogenous peptidome along with bioinformatics analysis to identify and evaluate potential biomarkers for HCC. Serum samples from 40 subjects, comprising 20 HCC cases and 20 patients with liver cirrhosis (CIRR), were analyzed. Among 2568 endogenous peptides, 67 showed significant differential expression between the HCC vs CIRR. Further analysis revealed three endogenous peptides (VMHEALHNHYTQKSLSLSPG, NRFTQKSLSLSPG, and SARQSTLDKEL) that showed far better performance compared to AFP in terms of area under the receiver operating characteristic curve (AUC), showcasing their potential as biomarkers for HCC. Additionally, endogenous peptide IAVEWESNGQPENNYKT that belongs to the precursor protein Immunoglobulin heavy constant gamma 4 was detected in 100% of the HCC group and completely absent in the CIRR group, suggesting a promising diagnostic biomarker. Gene ontology and pathway analysis revealed the potential involvement of these dysregulated peptides in HCC. These findings provide valuable insights into the molecular basis of HCC and may contribute to the development of improved diagnostic methods and therapeutic targets for HCC.

Keywords: biomarker; endogenous peptides; liver cancer; nano-LC-MS/MS; serum.

Figure 1

Workflow of nano-LC-MS/MS-based endogenous peptide analysis for HCC biomarker discovery.

Figure 2

Score plots of PCA (a) and PLS-DA (b) depicting HCC (red) and CIRR (green) cohorts.

Figure 3

(a) Volcano plot highlighting significantly up- and down-regulated endogenous peptides with |FC| > 1.5 and FDR < 0.05. (b) Venn diagram of 1903 endogenous peptides detected in more than 70% samples in each group (CIRR and HCC). (c) Heatmap of serum endogenous peptides differently expressed in HCC vs CIRR.

Figure 4

Box plots for eight endogenous peptides upregulated in HCC vs CIRR compared to alpha-fetoprotein (AFP) measured in the clinic having p-value <0.0001.

Figure 5

Box plots for eight endogenous peptides downregulated in HCC vs CIRR having p-value <0.0001.

Figure 6

ROC curves of the top five endogenous peptides having the highest AUC values in comparison with those of AFP.

Figure 7

(a) Canonical pathways associated with the precursor proteins listed in Table 2. The horizontal bars indicate the negative logarithm function of the overlap p-value, which represents the statistical significance of the pathway’s association with the specified proteins. (b) A network of the 15 upstream regulator molecules significantly associated with the genes encoding the proteins listed in Table 2.