Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas 66047, United States.
J Am Soc Mass Spectrom. 2021 Feb 3;32(2):436-443. doi: 10.1021/jasms.0c00317. Epub 2020 Dec 10.
Uromodulin, also known as the Tamm-Horsfall protein or THP, is the most abundant protein excreted in human urine. It is associated with the progression of kidney diseases; therefore, changes in the glycosylation profile of this protein could serve as a potential biomarker for kidney health. The typical glycomics analysis approaches used to quantify uromodulin glycosylation involve time-consuming and tedious glycoprotein isolation and labeling steps, which limit their utility in clinical glycomics assays, where sample throughput is important. Herein, we introduce a radically simplified sample preparation workflow, with direct ESI-MS analysis, enabling the quantification of N-linked glycans that originate from uromodulin. The method omits any glycan labeling steps but includes steps to reduce the salt content of the samples, thereby minimizing ion suppression. The method is effective for quantifying subtle glycosylation differences of uromodulin samples derived from different biological states. As a proof of concept, glycosylation from samples that differ by pregnancy status were shown to be differentiable.
尿调蛋白，也被称为 Tamm-Horsfall 蛋白或 THP，是人体尿液中排泄量最多的蛋白质。它与肾脏疾病的进展有关；因此，这种蛋白质的糖基化谱的变化可以作为肾脏健康的潜在生物标志物。用于定量尿调蛋白糖基化的典型糖组学分析方法涉及耗时且繁琐的糖蛋白分离和标记步骤，这限制了它们在临床糖组学分析中的应用，因为样品通量很重要。在此，我们引入了一种简化的样品制备工作流程，直接进行 ESI-MS 分析，从而能够定量源自尿调蛋白的 N-连接聚糖。该方法省略了任何聚糖标记步骤，但包括降低样品盐含量的步骤，从而最大程度地减少离子抑制。该方法可有效定量源自不同生物学状态的尿调蛋白样品的细微糖基化差异。作为概念验证，显示出不同妊娠状态的样品的糖基化是可区分的。
