Zhao Yang, Zhang Yong, Meng Bo, Luo Mengqi, Li Guisen, Liu Fang, Chang Cheng, Dai Xinhua, Fang Xiang
Mass Spectrometry Engineering Technology Research Center, Center for Advanced Measurement Science, National Institute of Metrology, Beijing, 102206 China.
Department of Nephrology, Institutes for Systems Genetics, West China Hospital, Sichuan University, Chengdu, 610041 China.
Phenomics. 2024 Apr 10;4(3):213-226. doi: 10.1007/s43657-023-00150-w. eCollection 2024 Jun.
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.
The online version contains supplementary material available at 10.1007/s43657-023-00150-w.
已表明人血浆免疫球蛋白γ分子（IgG）的位点特异性N-糖基化变化可调节免疫反应，并可作为准确诊断各种疾病的潜在生物标志物。然而，在大规模临床样本中准确量化完整的N-糖肽仍然是一项挑战，慢性肾脏病（CKD）患者血浆IgG的定量N-糖基化尚未得到研究。在本研究中，我们提出了一种新型的综合完整N-糖肽定量流程（称为GlycoQuant），它结合了我们最近开发的质谱碎裂方法（EThcD-sceHCD）和一个完整N-糖肽批量定量软件工具（升级版PANDA v.1.2.5）。我们在一小时内从58名健康对照（HC）、48名膜性肾病（MN）患者和35名IgA肾病（IgAN）患者中纯化并消化了人血浆IgG。然后，我们使用EThcD-sceHCD-MS/MS对消化后的肽段进行非富集分析，该方法提供了更高的谱图质量和更大的鉴定深度。使用升级版PANDA，我们对IgG进行了位点特异性N-糖基化定量。几个定量的完整N-糖肽不仅能将CKD与HC区分开来，还能区分不同类型的CKD（MN和IgAN），并可作为肾小管功能的准确诊断工具。此外，我们通过重新分析我们之前鉴定的细胞、尿液、血浆和组织样本中的完整N-糖肽，证明了该流程对复杂样本的适用性。我们相信，该流程可应用于大规模临床N-糖蛋白质组学研究，有助于发现新的糖基化生物标志物。
在线版本包含可在10.1007/s43657-023-00150-w获取的补充材料。
