Nitrogen-modified reduced graphene oxide for serum enrichment of N-glycans and MALDI-TOF MS-based identification of HCC biomarkers

Protein N-glycosylation, as one of the most crucial post-translational modifications, plays a significant role in various biological processes. The structural alterations of N-glycans are closely associated with the onset and progression of numerous diseases. Therefore, the precise and specific identification of disease-related N-glycans in complex biological samples is invaluable for understanding their involvement in physiological and pathological processes, as well as for discovering clinical diagnostic biomarkers. However, protein N-glycosylation suffers from microscopic heterogeneity and low abundance in biological systems, leading to N-glycopeptide signals being overshadowed by those of their non-glycosylated counterparts during mass spectrometry (MS) analysis. Consequently, there is an urgent demand for the development of novel methods for highly efficient N-glycan enrichment. In this study, we introduced a novel hydrophilic nanomaterial, nitrogen-modified reduced graphene oxide (N-rGO), tailored for this purpose, which was formed by a condensation reaction between the amino groups of rGO and the carboxyl groups of Fmoc-Photo-Linker. Compared to other enrichment materials, N-rGO not only supports efficient N-glycans enrichment via hydrophilic interaction (HILIC), but also serves as an effective matrix for direct MALDI-TOF MS analysis combined with DHB, thereby avoiding sample loss during N-glycans release. 76 and 81 serum N-glycans were obtained from 3 healthy individuals and 3 hepatocellular carcinoma (HCC) patients. Notably, relative quantification of serum N-glycans between 20 patients and 20 healthy controls showed significant expression differences, such as H5N4F1S1, H6N5F1, H5N4S2, H5N4F2S1 and H5N5F1S1, indicating the potential of N-rGO for biomarker discovery.