Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan; Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Division of Nephrology, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; Division of Nephrology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan; Taipei Medical University-Research Center of Urology and Kidney (TMU-RCUK), Taipei Medical University, Taipei, Taiwan.
J Chromatogr A. 2022 Dec 6;1685:463589. doi: 10.1016/j.chroma.2022.463589. Epub 2022 Oct 22.
Immunoglobulin A nephropathy (IgAN) is a highly prevalent autoimmune renal disease. Human IgA1 with galactose deficiency in the hinge region (HR) has been identified as an autoantigen for this disease. Therefore, analyzing IgA1 HR glycoforms in biofluids is important for biomarker discovery. Herein, an analytical method that includes one-pot sample preparation with unbiased plasma IgA purification, dual internal standard addition, and sensitive ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectroscopy (UHPLC-QqQ-MS/MS) was developed. Targeted O-glycopeptides detection was performed in pooled plasma with the validation of theoretical retention times, enzymatic treatment outcomes, product ion scans, and signal repeatability. A total of 42 IgA1 O-glycopeptides with N-acetylgalactosamines, galactoses, and sialic acids were determined from 8 µL of plasma. The newly developed method was applied to plasma samples from 16 non-IgAN controls and 19 IgAN patients. Comparing the 42 targets, 16 IgA1 HR O-glycopeptides were statistically different between the two groups (p<0.05). Decreased sialylation was identified in the IgA1 hinge region of IgAN patients, which was also correlated with the estimated glomerular filtration rate (eGFR). The developed method is sensitive and precise and can be used to identify plasma biomarkers for IgA nephropathy.
免疫球蛋白 A 肾病（IgAN）是一种高度流行的自身免疫性肾脏疾病。在铰链区（HR）缺乏半乳糖的人 IgA1 已被确定为此疾病的自身抗原。因此，分析生物体液中的 IgA1 HR 糖型对于生物标志物的发现很重要。本文开发了一种分析方法，包括一锅法样品制备，采用无偏的血浆 IgA 纯化、双重内标添加和灵敏的超高效液相色谱-三重四极杆串联质谱（UHPLC-QqQ-MS/MS）。在合并的血浆中进行靶向 O-糖肽检测，通过理论保留时间、酶处理结果、产物离子扫描和信号重复性验证。从 8µL 血浆中确定了 42 种带有 N-乙酰半乳糖胺、半乳糖和唾液酸的 IgA1 O-糖肽。新开发的方法应用于 16 名非 IgAN 对照者和 19 名 IgAN 患者的血浆样本。比较这 42 个靶标，两组间有 16 种 IgA1 HR O-糖肽具有统计学差异（p<0.05）。IgAN 患者 IgA1 铰链区的唾液酸化减少，这也与估计的肾小球滤过率（eGFR）相关。所开发的方法灵敏且精确，可用于鉴定 IgA 肾病的血浆生物标志物。
