PMID: 25153361

 

    Legend: Gene, Sites

Title : Confident assignment of site-specific glycosylation in complex glycoproteins in a single step

Abstract :
  1. A glycoprotein may contain several sites of glycosylation, each of which is heterogeneous
  2. As a consequence of glycoform diversity and signal suppression from nonglycosylated peptides that ionize more efficiently, typical reversed-phase LC-MS and bottom-up proteomics database searching workflows do not perform well for identification of site-specific glycosylation for complex glycoproteins
  3. We present an LC-MS system for enrichment, separation, and analysis of glycopeptides from complex glycoproteins (>4 N-glycosylation sequons) in a single step
  4. This system uses an online HILIC enrichment trap prior to reversed-phase C18-MS analysis
  5. We demonstrated the effectiveness of the system using a set of glycoproteins including human transferrin (2 sequons), human alpha-1-acid glycoprotein (5 sequons), and influenza A virus hemagglutinin (9 sequons)
  6. The online enrichment renders glycopeptides the most abundant ions detected, thereby facilitating the generation of high-quality data-dependent tandem mass spectra
  7. The tandem mass spectra exhibited product ions from both glycan and peptide backbone dissociation for a majority of the glycopeptides tested using collisionally activated dissociation that served to confidently assign site-specific glycosylation
  8. We demonstrated the value of our system to define site-specific glycosylation using a hemagglutinin containing 9 N-glycosylation sequons from a single HILIC- C18-MS acquisition
Output (sent_index, trigger, protein, sugar, site):
  • 0. glycoproteins, , glycoproteins, -, -
  • 1. glycoprotein, , glycoprotein, -, -
  • 1. glycosylation, , -, -, sites
  • 2. glycoproteins, , glycoproteins, -, -
  • 2. nonglycosylated, , -, -, peptides
  • 3. N-glycosylation, , -, -, -
  • 3. glycopeptides, , -, -, glycopeptides
  • 3. glycopeptides, , glycoproteins, -, -
  • 3. glycopeptides, , glycoproteins, -, glycopeptides
  • 3. glycoproteins, , glycoproteins, -, -
  • 5. glycoprotein, , glycoprotein (5, -, -
  • 5. glycoproteins, , glycoprotein (5, -, -
  • 5. glycoproteins, , glycoproteins, -, -
  • 5. glycoproteins, , hemagglutinin, -, -
  • 5. glycoproteins, , transferrin, -, -
  • 6. glycopeptides, , -, -, glycopeptides
  • 7. glycopeptides, , -, -, glycopeptides
  • 8. N-glycosylation, , -, -, -
Output(Part-Of) (sent_index, protein, site):
  • 1. glycoprotein, sites
  • 3. glycoproteins, glycopeptides
  • 5. alpha-1-acid, -
  • 8. hemagglutinin, -
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX
C18 27241 4,8
transferrin 7018 5