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Sequential phosphoproteomics and N-glycoproteomics of plasma-derived extracellular vesicles

ABSTRACT: Extracellular vesicles (EVs) are increasingly recognized as important cargos for intercellular communication and promising sources for less invasive biomarker discovery. As the state of protein post-translational modifications (PTMs) such as phosphorylation and glycosylation can be a key determinant of cellular physiology such as early stage cancer, comprehensive characterization of protein PTMs in EVs are highly valuable, but significant challenges remain. Recently we demonstrated the feasibility to isolate and identify phosphoproteins and glycoproteins in EVs from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides and 1,500 unique glycopeptides in EVs isolated from small volumes of plasma samples. Using label-free quantitative phospho- and glyco-proteomics, we identified a number of phosphoproteins and glycoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer. Here we present an updated workflow to sequentially isolate phosphopeptides and N-glycopeptides, enabling multiple PTM analyses with the same clinical samples and presenting intriguing functional molecules for further validation.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hillary Andaluz

LAB HEAD: W. Andy Tao

PROVIDER: PXD013893 | Pride | 2019-09-25

REPOSITORIES: Pride

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			Action	DRS
	050319-EVs-plasma-phospho-alone-1.raw	Raw
	050319-EVs-plasma-phospho-alone-2.raw	Raw
	050319-EVs-plasma-phospho-alone-3.raw	Raw
	050319-EVs-plasma-phospho-sequential-1.raw	Raw
	050319-EVs-plasma-phospho-sequential-2.raw	Raw

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Project description:Background Plasma proteins can be modified by addition of sugar residues by non-enzymatic glycation as well as glycosylation. While glycation is a hallmark of hyperglycemia, glycosylation is a complex, enzyme-catalyzed post-translational modification by heterogeneous sugar chains and is present on a majority of plasma proteins. N-linked glycosylation occurs on asparagine residues occurring predominantly on a canonical N-glycosylation motif (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported to be glycosylated less frequently. Albumin is the most abundant protein in human plasma whose glycation has been implicated in end-organ damage in advanced diabetes mellitus, and as such, has both diagnostic and therapeutic implications. However, albumin has long been regarded as a non-glycosylated protein owing to the absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated with two possible attached glycans. Methods To investigate N-linked glycosylation of abundant serum proteins in greater detail, we passed healthy donor samples through a multiple affinity removal spin (MARS14) column followed by trypsin digestion of bound proteins and subsequent glycopeptide enrichment by size-exclusion chromatography (SEC) or mixed-mode anion-exchange (MAX), in parallel. Global analysis of intact glycopeptides was performed by LC-MS/MS to evaluate potential glycosylation at canonical as well as non-canonical sites. PNGase F treatment was carried out to confirm N-linked glycosylation at non-canonical sites Asn-X-Cys/Val. Glycopeptides on albumin at non-canonical sites were further characterized by MS3 fragmentation from immunoprecipitated albumin to confirm the attached glycans. To assess if the observed glycosylation was a general phenomenon, targeted analysis using parallel reaction monitoring (PRM) was carried out on an independent set of twenty human serum samples. Finally, to test whether albumin glycosylation is unique to human albumin or is a general phenomenon, bovine and rabbit serum albumins were subjected to MAX chromatography and LC-MS/MS analysis. Results We report that human albumin is modified by N-linked glycosylation at two sites, Asn68(-Glu-Val) and Asn123-(Glu-Cys), both of which occur in non-canonical N-glycosylation motifs. We detected N-glycopeptides at both sites bearing four complex mono- and bi-antennary N-linked glycans, with Hex5HexNAc4NeuAc2 and Hex5HexNAc4NeuAc1 being the most abundant glycans. These findings were validated by analyzing deglycosylated peptides and MS3-based fragmentation of glycopeptides. Glycosylation at both sites was confirmed by targeted MS in twenty donor samples. Finally, we report that the Asn residue on bovine and rabbit serum albumins that corresponds to the highly conserved Asn123 in human albumin is also N-glycosylated with complex sialylated glycans similar to those observed in human albumin. Conclusions Albumin is a conserved glycoprotein with multiple N-linked glycosylation sites. This glycosylation of albumin has potential functional implications and applications in medicine.

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Sequential phosphoproteomics and N-glycoproteomics of plasma-derived extracellular vesicles

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