PMID: PMC3617342-1-4

 

    Legend: Gene, Sites

Title : The Endothelial Glycoproteome

Abstract :
  1. Among the 1252 identified proteins were 253 extracellular or plasma membrane proteins (approximately 20%) related to cell adhesion, blood coagulation, hemostasis, signaling transduction, and protein transportation, of which 166 were known glycoproteins (Table I)
  2. To further characterize this subproteome, we employed a glycoproteomics approach
  3. Secreted proteins were precipitated and digested with trypsin, and tryptic peptides were labeled with TMT0 to increase their charge state prior to enrichment by means of zwitterionic hydrophilic interaction liquid chromatography purification (24)
  4. For glycosite identification, an indirect and a direct strategy were pursued (Fig. 2A): (i) digestion with PNGase F in the presence of 18O water to label the conversion of asparagine to aspartic acid upon the removal of N-glycans, and (ii) alternating HCD and ETD (HCD-alt-ETD) or HCD-product-dependent ETD ( HCD-pd-ETD) fragmentation on an Orbitrap Elite MS (24)
  5. There was little overlap in the numbers of glycopeptides (Fig. 2B) and glycosylation sites (Fig. 2C) identified via the direct ( HCD-ETD) and the indirect ( PNGase F + H218O) methods
  6. Better agreement was observed at the protein level (Fig. 2D)
  7. With the indirect ( PNGase F + H218O) method, 27 peptides were identified with N[+2.99] modification at non-consensus sequence , out of 1139 total identified peptides with N[+2.99]
  8. This anomaly rate of 2.4% (27/1139) combines the rate of false identifications and the rate of chance deamidations in 18O water that were not in the consensus sequence of glycosylation (i.e. N-X(not P)-S/T)
  9. All glycopeptides identified are listed in Table II and supplemental Table S4
  10. Three spectra (full MS, HCD , and ETD) from a neuronal cell adhesion molecule (UniProt accession number Q92823) (AA - 222FNHTQTIQQK231) are presented in Fig. 3
  11. For the same samples, HCD-pd-ETD revealed 28 known, 25 potential, and 16 novel glycosylation sites based on 209 identified spectra; HCD-alt-ETD revealed 20 known, 32 potential, and 14 novel glycosylation sites from 110 identified spectra
  12. The HCD-alt-ETD method selected mostly precursors with higher intensities, higher charge, and smaller m/z (Fig. 4A)
  13. Several large glycopeptides were detected via only HCD-alt-ETD , and more low-abundant glycopeptides were detected via HCD-pd-ETD
  14. There was limited overlap in the identified glycopeptides but better agreement in the protein level (Fig. 4B)
  15. Among the 319 total glycopeptides identified in the conditioned media, 31 were attached with a trimannosyl core (-HexNAc2Hex3) or truncated core (-HexNAc2Hex) , 50 with high mannose (-HexNAc2Hex4–9) , and 238 with complex/hybrid glycans
  16. Notably, HCD-pd-ETD detected almost twice as many complex/hybrid glycoforms as HCD-alt-ETD (Fig. 4C)
Output (sent_index, trigger, protein, sugar, site):
  • 1. glycoproteins, , glycoproteins, -, -
  • 11. glycosylation, , -, -, sites
  • 13. glycopeptides, , -, -, glycopeptides
  • 14. glycopeptides, , -, -, glycopeptides
  • 15. glycopeptides, , -, -, glycopeptides
  • 4. glycosite, , -, -, glycosite
  • 5. glycopeptides, , -, -, glycopeptides
  • 5. glycosylation, , -, -, sites
  • 9. glycopeptides, , -, -, glycopeptides
Output(Part-Of) (sent_index, protein, site):
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX