N-linked (N-) Glycoproteomics of Urimary Exosomes*

Abstract

Epithelial cells lining the urinary tract secrete urinary exosomes (40 -100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk.Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS.We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 Nglycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of Nglycoproteome of urinary exosomes. Molecular & Cellular Proteomics

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation

“…Taken together, our glycomics study demonstrated the prevalence of complex N -glycans in all particle subsets with relatively high levels of sialylation, consistent with previous findings of complex N -glycans and sialoglycoproteins in tumor microvesicles/exosomes 25-27 . Furthermore, our study revealed differences in N- glycan composition and structures among exomeres, Exo-S, and Exo-L.…”

Current state of the art for enhancing urine biomarker discovery

“…examined urinary exosome glycosylation patterns because the exosome glycosylation pattern influences exosome targeting and uptake [98]. 37 glycoproteins were identified in urinary exosomes by comparing the urinary exosome glycan structures to a database rather than through iterative de novo glycan structure analysis [98]. Glycoproteins were digested with trypsin prior to enrichment by Sambucus nigra agglutinin (SNA) affinity chromatography or size exclusion chromatography.…”

“…SNA binds preferentially to sialic acid attached to terminal galactose in α-2,6 and to a lesser degree, α-2,3 linkages. The glycoprotein enriched fractions were analyzed using collision induced dissociation-Tandem MS [98]. From these glycoproteins, 126 N -glycopeptides from 51 N -glycosylation sites were characterized [98].…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…The different programs utilize various strategies to identify N-glycopeptides including the use of characteristic Y 1 ions, oxonium ions, B/Y-and C/Z-type glycan fragment ion series, and b/y-and c/z-type peptide fragment ions (116,117). In order to limit the search space and enhance the detailed structural knowledge of the glycans encountered in a given glycoproteome, several studies have performed parallel N-glycome profiling (21,40,88,105,106,108,162). This so-called 'glycomics-assisted glycoproteomics' approach can also be complemented with quantitative proteome and de-N-glycoproteome profiling (mapping of formerly N-glycosylated peptides) (21, 46, 88, 103, 105-108, 135, 159, 162), which reduce the search space even further and provide supporting evidence to pinpoint the exact mechanism(s) driving the observed glycoproteome alterations.…”

“…Other new enrichment methods of interest include the metabolic incorporation of N-azido sugars into N-glycopeptides to facilitate their specific enrichment and detection (84,85). The selective precipitation of glycopeptides by acetone (86), use of size exclusion chromatography (87,88) and the combined use of porous graphitized carbon (PGC) and reversed phase (RP) (89) and titanium dioxide (90) solid phase extraction (SPE) for efficient enrichment of glycopeptides and sialoglycopeptides, respectively, are also promising developments. However, common for most of these proof-of-principle methodology studies is the need for further validation to demonstrate their true potential in glycoproteomics.…”