Direct N-glycosylation profiling of urine and prostatic fluid glycoproteins and extracellular vesicles

Expressed prostatic secretions (EPS), also called post digital rectal exam urines, are proximal fluids of the prostate that are widely used for diagnostic and prognostic assays for prostate cancer. These fluids contain an abundant number of glycoproteins and extracellular vesicles secreted by the prostate gland, and the ability to detect changes in their N-glycans composition as a reflection of disease state represents potential new biomarker candidates. Methods to characterize these N-glycan constituents directly from clinical samples in a timely manner and with minimal sample processing requirements are not currently available. In this report, an approach is described to directly profile the N-glycan constituents of EPS urine samples, prostatic fluids and urine using imaging mass spectrometry for detection. An amine reactive slide is used to immobilize glycoproteins from a few microliters of spotted samples, followed by peptide N-glycosidase digestion. Over 100 N-glycan compositions can be detected with this method, and it works with urine, urine EPS, prostatic fluids, and urine EPS-derived extracellular vesicles. A comparison of the N-glycans detected from the fluids with tissue N-glycans from prostate cancer tissues was done, indicating a subset of N-glycans present in fluids derived from the gland lumens. The developed N-glycan profiling is amenable to analysis of larger clinical cohorts and adaptable to other biofluids

Metabolic Markers and Association of Biological Sex in Lupus Nephritis

Lupus nephritis (LN) is a serious complication for many patients who develop systemic lupus erythematosus, which primarily afflicts women. Our studies to identify biomarkers and the pathogenic mechanisms underlying LN will provide a better understanding of disease progression and sex bias, and lead to identification of additional potential therapeutic targets. The glycosphingolipid lactosylceramide (LacCer) and N-linked glycosylated proteins (N-glycans) were measured in urine and serum collected from LN and healthy control (HC) subjects (10 females and 10 males in each group). The sera from the LN and HC subjects were used to stimulate cytokine secretion and intracellular Ca2+ flux in female- and male-derived primary human renal mesangial cells (hRMCs). Significant differences were observed in the urine of LN patients compared to HCs. All major LacCers species were significantly elevated and differences between LN and HC were more pronounced in males. 72 individual N-glycans were altered in LN compared to HC and three N-glycans were significantly different between the sexes. In hRMCs, Ca2+ flux, but not cytokine secretion, was higher in response to LN sera compared to HC sera. Ca2+ flux, cytokine secretion, and glycosphingolipid levels were significantly higher in female-derived compared to male-derived hRMCs. Relative abundance of some LacCers and hexosylceramides were higher in female-derived compared to male-derived hRMCs. Urine LacCers and N-glycome could serve as definitive LN biomarkers and likely reflect renal disease activity. Despite higher sensitivity of female hRMCs, males may experience greater increases in LacCers, which may underscore worse disease in males. Elevated glycosphingolipid metabolism may poise renal cells to be more sensitive to external stimuli