A Novel Integrated Pipeline for Site-Specific Quantification of N-glycosylation

The site-specific N-glycosylation changes of human plasma immunoglobulin gamma molecules (IgGs) have been shown to modulate the immune response and could serve as potential biomarkers for the accurate diagnosis of various diseases. However, quantifying intact N-glycopeptides accurately in large-scale clinical samples remains a challenge, and the quantitative N-glycosylation of plasma IgGs in patients with chronic kidney diseases (CKDs) has not yet been studied. In this study, we present a novel integrated intact N-glycopeptide quantitative pipeline (termed GlycoQuant), which combines our recently developed mass spectrometry fragmentation method (EThcD-sceHCD) and an intact N-glycopeptide batch quantification software tool (the upgraded PANDA v.1.2.5). We purified and digested human plasma IgGs from 58 healthy controls (HCs), 48 patients with membranous nephropathy (MN), and 35 patients with IgA nephropathy (IgAN) within an hour. Then, we analyzed the digested peptides without enrichment using EThcD-sceHCD-MS/MS, which provided higher spectral quality and greater identified depth. Using upgraded PANDA, we performed site-specific N-glycosylation quantification of IgGs. Several quantified intact N-glycopeptides not only distinguished CKDs from HCs, but also different types of CKD (MN and IgAN) and may serve as accurate diagnostic tools for renal tubular function. In addition, we proved the applicability of this pipeline to complex samples by reanalyzing the intact N-glycopeptides from cell, urine, plasma, and tissue samples that we had previously identified. We believe that this pipeline can be applied to large-scale clinical N-glycoproteomic studies, facilitating the discovery of novel glycosylated biomarkers.