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.