PMID: 26598643

 

    Legend: Sugar

Title : Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins

Abstract :
  1. Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins , e.g. in the context of antigenicity and disease progression
  2. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins
  3. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation
  4. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins
  5. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme ( Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans , which represent the vast majority of O-glycans on human blood plasma proteins
  6. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode
  7. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode
  8. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation
  9. Spectra were annotated by database searches as well as manually
  10. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins
  11. Strikingly, also 11 novel O-glycosylation sites and regions were identified
  12. In total 23 O-glycosylation sites could be pinpointed
  13. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context
  14. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen)
  15. The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships
  16. The data were deposited to ProteomeXchange PXD003270