Evaluation of serum phosphopeptides as potential biomarkers of gastric cancer

Abstract

Gastric cancer (GC) is the fourth most commonly diagnosed cancer and the third leading cause of cancer-related deaths. Most GC deaths can be prevented by early diagnosis. However, biomarkers with high sensitivity and specificity are rare. Comprehensive serum phosphopeptides analysis may lead to the discovery of novel biomarkers for GC. In this work, we report a mass spectrometric strategy for the evaluation of serum phosphopeptides separated and enriched by ZrAs-Fe₃O₄@SiO₂ nanoparticles. Four endogenous phosphopeptides in sera generated by degradation of fibrinogen were synthesized with light- and heavy-glycine residues, respectively, as external/internal standards, and used to gain multi-point standard calibration curves for absolute quantification of phosphopeptides by LC-ESI-MS following ZrAs-Fe₃O₄@SiO₂ enrichment. The ESI-MS signal ratios of the four pairs of light-/heavy-phosphopeptide standards captured by ZrAs-Fe₃O₄@SiO₂ from aqueous solutions are linearly correlated with the molar ratios of the “light” to “heavy” phosphopeptides over the range of 0.05–5 μM with an r² of up to 0.998 and a slope of close to 1. The recovery of the four phosphopeptides spiked at low, medium and high levels in human sera were 94.7–107.5% with RSDs in the range of 0.6–8.2%. The validated method was utilized to measure the phosphopeptide levels of serum samples from 30 healthy persons and 60 GC patients. Receiver operating characteristic (ROC) analysis shows that one phosphopeptide (F3: DpSGEGDFLAEGGGVR) yields high sensitivity and specificity of 90.0% and 96.7%, respectively, in the validation set for discriminating GC patients from healthy controls. Overall, 53 of 60 GC cases and 29 of 30 controls were correctly classified, including eight of nine GC patients at stage I. These results suggest that F3 in sera may be a potential biomarker for GC diagnosis, particularly for early stage cases.