posted on 2021-03-02, 14:36authored byHaiying Li, Stephen A. Kostel, Shannon E. DiMartino, Ali Hashemi Gheinani, John W. Froehlich, Richard S. Lee
The
glycoprotein uromodulin (UMOD) is the most abundant protein
in urine, and N-glycans are critical for many biological
functions of UMOD. Comprehensive glycan profiling of UMOD provides
valuable information to understand the exact mechanisms of glycan-regulated
functions. To perform comprehensive glycosylation analysis of UMOD
from urine samples with limited volumes, we developed a streamlined
workflow that included UMOD isolation from 5 mL of urine from 6 healthy
adult donors (3 males and 3 females) and a glycosylation analysis
using a highly sensitive and reproducible nanoLC-MS/MS based glycomics
approach. In total, 212 N-glycan compositions were
identified from the purified UMOD, and 17% were high-mannose glycans,
2% were afucosylated/asialylated, 3% were neutral fucosylated, 28%
were sialylated (with no fucose), 46% were fucosylated and sialylated,
and 4% were sulfated. We found that isolation of UMOD resulted in
a significant decrease in the relative quantity of high-mannose and
sulfated glycans with a significant increase of neutral fucosylated
glycans in the UMOD-depleted urine relative to the undepleted urine,
but depletion had little impact on the sialylated glycans. To our
knowledge, this is the first study to perform comprehensive N-glycan profiling of UMOD using nanoLC-MS/MS. This analytical
workflow would be very beneficial for studies with limited sample
size, such as pediatric studies, and can be applied to larger patient
cohorts not only for UMOD interrogation but also for global glycan
analysis.