Clinically Viable Assay for Monitoring Uromodulin Glycosylation

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.

尿调节素（Uromodulin），又称Tamm-Horsfall蛋白（THP），是人体尿液中含量最高的排泄蛋白。其与肾脏疾病的进展密切相关，因此该蛋白的糖基化谱变化可作为评估肾脏健康状态的潜在生物标志物。当前用于定量尿调节素糖基化的典型糖组学分析方法，需经历耗时且繁琐的糖蛋白分离与标记步骤，这限制了其在临床糖组学检测中的应用——临床检测对样本通量具有较高要求。本文提出了一种大幅简化的样本制备流程，结合直接ESI-MS分析，可实现源自尿调节素的N-连接聚糖的定量检测。该方法无需任何聚糖标记步骤，但包含降低样本盐含量的环节，从而最大程度减轻离子抑制效应。该方法可有效定量不同生物学状态下尿调节素样本的细微糖基化差异。作为概念验证，研究证实妊娠状态不同的样本间的糖基化特征可被有效区分。