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.

尿调蛋白（Uromodulin），又称Tamm-Horsfall蛋白（THP），是人体尿液中含量最高的分泌蛋白。该蛋白与肾脏疾病的进展密切相关，因此其糖基化谱的变化可作为评估肾脏健康状况的潜在生物标志物。传统用于定量尿调蛋白糖基化的糖组学（glycomics）分析方法，需历经耗时繁琐的糖蛋白分离与标记步骤，这极大限制了其在样本通量至关重要的临床糖组学检测中的应用价值。本文介绍了一种大幅简化的样本前处理流程，结合直接电喷雾电离质谱（ESI-MS）分析，可实现源自尿调蛋白的N-连接聚糖（N-linked glycans）的定量检测。该方法无需任何糖基标记步骤，仅需对样本进行脱盐处理，从而最大限度降低离子抑制（ion suppression）效应。该方法可有效定量不同生理状态下尿调蛋白样本的细微糖基化差异。作为概念验证（proof of concept），本研究证实妊娠状态不同的样本其糖基化特征具有可区分性。