Accurate profiles of IgA1 O-glycan heterogeneity in IgA nephropathy

描述（由申请人提供）：IgA 肾病 (IgAN) 是世界上最常见的肾小球肾炎形式，也是终末期肾病的主要原因。尽管其发病机制尚不清楚，但多个实验室的报告表明，循环和肾脏沉积物中存在的异常糖基化 IgA1 发挥着关键作用。该 IgA1 重链铰链区 (HR) 中的一些 3-5 O 连接聚糖缺乏半乳糖 (Gal)。因此，在 IgAN 患者中，IgA1 O 连接聚糖被截短，末端为 N 乙酰半乳糖胺 (GalNAc) 或唾液酸化 GalNAc。多种方法（聚糖特异性凝集素结合测定、MALDI-TOF MS 方法和聚糖成分色谱分析）已检测到正常健康对照和 IgAN 患者的 IgA1 O 聚糖群体之间存在明显差异。然而，正常和缺乏 Gal 的聚糖的附着位点仍有待确定。因此，需要一种能够提供有关每个样品中 O-糖肽群的详细结构信息的方法。傅里叶变换离子回旋共振质谱 (FT-ICR MS) 在鉴定生物分子（包括从血清 IgA1 酶促释放的 IgA1 O-糖肽群）方面提供无与伦比的质量精度。我们最近通过电子捕获解离 (ECD) FT-ICR 串联 MS 定位（首次通过直接方法）三种不同 IgA1 HR 糖肽中的多个 O-聚糖链。这种方法使我们能够检验我们的假设，即与正常健康人和疾病对照相比，IgAN 患者血清 IgA1 O-聚糖群体在组成和附着位点方面存在差异，此外，这些差异可以作为疾病的标志物并定义病理特征。为了在 IgAN 患者中建立准确的 IgA1 糖型谱，我们提出以下建议： 1. 提供 IgAN 住院患者血清 IgA1 糖型的 FT-ICR 准确质量谱以及正常健康人和疾病对照的基线谱。 2. 通过 ECD FT-ICR 串联质谱法定位 IgAN 患者、正常健康对照和其他形式肾小球肾炎患者的个体血清 IgA1 糖型中 O 聚糖附着位点。我们对总共 115 个样本（45 个 IgAN、25 个狼疮性肾炎样本和 45 个健康对照）进行了这项分析。这些研究将提供有关 IgAN 中发现的异常糖基化模式的详细结构信息，深入了解该疾病的发病机制，并可能为 IgAN 的诊断提供一种新的非侵入性方法。公共健康相关性：这个为期两年的项目旨在利用新型质谱技术来确定 IgA 肾病 (IgAN) 患者血清 IgA1 异常 O-糖基化位点。这只能在了解正常健康对照和疾病对照中 IgA1 O-糖基化位点的背景下才能完成。该结果将提供对 IgAN 病理学的深入了解，并可能提供一种非侵入性的诊断该疾病的方法。

DESCRIPTION (provided by applicant): IgA nephropathy (IgAN) is the most common form of glomerulonephritis in the world and is a leading cause of end-stage renal disease. Although the mechanisms of its pathogenesis remain unclear, reports from several laboratories indicate a key role for an aberrantly glycosylated IgA1 present in the circulation and renal deposits. This IgA1 is galactose (Gal)-deficient in some of its 3-5 O-linked glycans in the hinge region (HR) of the heavy chain. Thus, in patients with IgAN, the IgA1 O-linked glycans are truncated, with terminal N acetylgalactosamine (GalNAc) or sialylated GalNAc. A variety of methods (glycan-specific lectin binding assays, MALDI-TOF MS methods, and chromatographic analysis of glycan composition) have detected clear differences between normal healthy controls' and IgAN patients' O-glycan populations in IgA1. However, the sites of attachment of normal and Gal-deficient glycans remain to be defined. Thus, a method is needed that can provide detailed structural information about the population of O-glycopeptides within each sample. Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) provides unmatched mass accuracy in the identification of biomolecules, including the population of IgA1 O-glycopeptides enzymatically released from serum IgA1. We have recently localized (for the first time by a direct method) the multiple O-glycan chains in three different IgA1 HR glycopeptides by electron capture dissociation (ECD) FT-ICR tandem MS. This methodology enables us to test our hypothesis that the populations of serum IgA1 O-glycans differ in composition and sites of attachment in patients with IgAN when compared to normal healthy and disease controls and, furthermore, that these differences can serve as markers of the disease and define the pathological features. To establish accurate profiles of IgA1 glycoforms in IgAN patients we propose the following: 1. Provide an FT-ICR accurate mass profile of serum IgA1 glycoforms found inpatients with IgAN as well as a baseline profile from normal healthy and disease controls. and 2. Localize sites of O-glycan attachment in individual serum IgA1 glycoforms from patients with IgAN, normal healthy controls, and patients with other forms of glomerulonephritis by ECD FT-ICR tandem mass spectrometry. We perform this analysis on a total of 115 samples (45 IgAN, 25 lupus nephritis, and 45 healthy controls). These studies will provide detailed structural information about the aberrant glycosylation patterns found in IgAN, give insights into the pathogenesis of this disease, and may provide a novel noninvasive method for diagnosis of IgAN. PUBLIC HEALTH RELEVANCE: This two year project seeks to use novel mass spectrometry techniques to define the sites of aberrant O-glycosylation of serum IgA1 in patients with IgA nephropathy (IgAN). This can only be done in context of understanding the sites of IgA1 O-glycosylation in normal healthy controls and disease controls. The results will provide insights into the pathology of the IgAN and may provide a non-invasive method of diagnosing th3disease.