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，是终末期肾脏疾病的主要原因。尽管其发病机制尚不清楚，但几个实验室的报告表明，循环和肾脏沉积物中存在异常糖基化的IgA1具有关键作用。该IgA1在重链的铰链区（HR）的3-5个o -连接聚糖中缺乏半乳糖（Gal）。因此，在IgAN患者中，IgA1 o连接的聚糖被截断，末端是N乙酰半乳糖胺（GalNAc）或唾液化的GalNAc。多种方法（聚糖特异性凝集素结合试验、MALDI-TOF质谱法和聚糖组成的色谱分析）已经检测到正常健康对照者和IgAN患者IgA1中o -聚糖群的明显差异。然而，正常和缺乏gal的聚糖的附着位点仍有待确定。因此，需要一种能够提供每个样品中o -糖肽种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）在鉴定生物分子方面提供了无与伦比的质量准确性，包括从血清IgA1酶促释放的IgA1 o糖肽的数量。我们最近通过电子捕获解离（ECD） FT-ICR串联质谱法定位了（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验证我们的假设，即与正常健康和疾病对照相比，IgAN患者血清IgA1 o -聚糖的组成和附着位点不同，此外，这些差异可以作为疾病的标志，并确定病理特征。为了在IgAN患者中建立准确的IgA1糖型图谱，我们提出以下建议：提供住院IgAN患者血清IgA1糖型的FT-ICR准确质量谱，以及正常健康和疾病对照的基线谱。和2。用ECD FT-ICR串联质谱法定位IgAN患者、正常健康对照者和其他形式肾小球肾炎患者血清IgA1糖型中o -聚糖附着位点。我们对总共115个样本（45个IgAN， 25个狼疮肾炎和45个健康对照）进行了这项分析。这些研究将提供关于IgAN异常糖基化模式的详细结构信息，深入了解该病的发病机制，并可能为IgAN的诊断提供一种新的无创方法。公共卫生相关性：这个为期两年的项目旨在使用新的质谱技术来确定IgA肾病（IgAN）患者血清IgA1异常o -糖基化的位点。这只能在了解正常健康对照和疾病对照中IgA1 o糖基化位点的情况下进行。该结果将为IgAN的病理学提供深入的见解，并可能提供一种诊断该疾病的非侵入性方法。

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.