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In-depth profiling of urinary N-glycome in diabetic kidney disease by ultrafast glycoprotein immobilization for glycan extraction (UltraGIG)

Yan Cai,Weifu Ren,Haiyan Wang,Qi Bian

Diabetic kidney disease (DKD) is a common and serious kidney-related complication of diabetic mellitus. Although albuminuria and estimated glomerular filtration rate (eGFR) are commonly used diagnostic biomarkers, they have limitations in the diagnosis of DKD due to the lack of specificity and sensitivity. Urinary N-glycans are emerging as novel biomarkers for predicting renal prognosis in DKD. However, most of the current N-glycan profiling methods for DKD were based on lectin affinity enrichment or hydrophilic interaction chromatography (HILIC) with optical detection, which provided limited N-glycome coverage in low throughput. Herein, we developed a novel N-glycan purification method, termed Ultrafast Glycoprotein Immobilization for Glycan extraction (UltraGIG), for in-depth profiling of urinary N-glycome in DKD using mass spectrometry (MS). In UltraGIG, proteins were rapidly (within 40 min) immobilized to resin with high efficiency (over 98%) through NHS-reactive chemistry , and then N-glycans were enzymatically released from the resin by PNGase F. Owing to good efficiency of immobilization of proteins to resin, the subsequent washing steps to remove proteins and other impurities could be easily performed. The UltraGIG showed good selectivity towards glycans (extracting N-glycans from the mixture of IgG and BSA at a 1:100 M ratio) and samples loss was minimized (detection sensitivity at fmol level). A total of 237 N-glycan compositions were identified from urine samples with DKD and healthy controls, which representing the largest data set of N-glycome from DKD. Compared to healthy controls, 3 N-glycans were up-regulated and 14 N-glycans were down-regulated in DKD patients. Collectively, UltraGIG offers a competitive sample purification method for in-depth analysis of urinary N-glycome by MS and provide new insights into biomarker study for the diagnosis of DKD.