Title : Caveats of the Approach
Abstract :
- In contrast to tryptic (glyco) peptides , Proteinase K generated peptides and glycopeptides cannot be predicted because of the broad cleavage specificity of the enzyme
- 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
- 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
- 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
- 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)
- 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)
- 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)
- 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):