PMID: PMC4739677-2-10

 

    Legend: Gene, Sites

Title : Caveats of the Approach

Abstract :
  1. In contrast to tryptic (glyco) peptides , Proteinase K generated peptides and glycopeptides cannot be predicted because of the broad cleavage specificity of the enzyme
  2. More importantly, though, is the reduced peptide length compared with a tryptic digest, as this can lead to an insufficient number of detected fragment ions to allow for unambiguous peptide identifications
  3. This problem can be even more intensified by the frequent occurrence of prolines within mucin-type O-glycopeptide sequences , as prolines can introduce additional sequence gaps during mass spectrometry-based peptide sequencing
  4. Also important to note is the increased search space of the search engine because of the use of a nonspecific enzyme , which results in an increased ambiguity with respect to the peptide identification (lower identification scores) and longer search times
  5. A confounding factor that relates to the ETD analysis is the predominance of charge state 2+ among the measured O-glycopeptide precursor ions, because ETD fragmentation is more efficient for precursor charge states greater than 2+ (86)
  6. The predominance of charge state 2+ can be explained by a lack of ionizable/basic amino acids (lack of Arg, Lys, His ) within the glycopeptides—a characteristic that can be linked to the broad-specific proteolytic digest by Proteinase K (87)
  7. Another caveat is related to the HILIC glycopeptide enrichment: this step was optimized to enrich O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, as they represent the vast majority of O-glycans on human blood plasma proteins (25)
  8. Hence, O-glycopeptides carrying bigger and thus more hydrophilic O-glycans, such as N-acetyl-lactosamine (LacNAc) extended mucin-type core-2 O-glycans, or O-glycopeptides carrying multiple mucin-type O-glycans, might elute in the subsequent washing phase of the HILIC fractionation and as a consequence cannot be found during the analysis
Output (sent_index, trigger, protein, sugar, site):
  • 1. glycopeptides, , -, -, peptides and glycopeptides
  • 3. O-glycopeptide, , -, -, O-glycopeptide sequences
  • 5. O-glycopeptide, , -, -, O-glycopeptide
  • 7. O-glycopeptides, , -, -, O-glycopeptides
  • 7. glycopeptide, , -, -, glycopeptide
  • 8. O-glycopeptides, , -, -, O-glycopeptides
Output(Part-Of) (sent_index, protein, site):
  • 1. Proteinase K, peptides and glycopeptides
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX