Title : Tandem MS ofGlycopeptides
Abstract :
- While the presence of oxoniumions from dissociation of glycosidic bonds is a useful feature, theformation of peptide backbone product ions is necessary for unambiguousassignment of the peptide sequence and glycan com position
- We thereforeinvestigated the extent to which the increased glycopeptide precursorion abundances obtained using HILIC-C18-MS improved the quality ofthe resulting glycopeptide tandem mass spectra
- Collisional dissociationusing typical conditions for peptide tandem MS results in formationof abundant ions from dissociation of the glycan with those from peptidebackbone dissociation often not detected
- In the interest of maximizingthe information produced from a CAD LC–MS/MS experiment, weinvestigated use of higher collision energies for glycopeptides
- Figures 3 and S-2 show examplesof glycopeptide tandem mass spectra from each of the three glycoproteinsstudied; collision energies were calculated as per the equations describedin Supporting Information Section S-1
- As expected, glycopeptide dissociation produced abundant oxonium ionsin the low m/z range that confirmedthe presence of monosaccharides in the precursor ion com position
- In addition, peptide backbone product ions were detected that enableddirect identification of the peptide
- This information increased confidencein true matches and decreased those in incorrect matches
- This wasimportant in cases where more than one theoretical glycopeptide matchedan observed mass value
- Complete peptide backbone or parts of thepeptide backbone could be sequenced for glycopeptide precursors , asshown in the annotated spectra
- Intact peptide and glycopeptides +saccharide (referred to as stub glycopeptide ) ions were also detectedin the higher m/z range of the spectrathat matched the masses of peptide with N-glycancore structures
- The presence of intact peptide or stub glycopeptideions significantly increased confidence of assignments over thosemade from MS-only data
- In the majority of LC–tandem mass spectra,ions corresponding to peptide backbone product ions plus a HexNAcresidue were detected, which confirmed the site of glycosylation,as shown in Supporting Information Figure S-2 and Tables 2 and S-2
- Although the relative abundanceof a glycopeptide precursor ionhad an effect on the quality of tandem MS by affecting the abilityto select the ion in a data-dependent LC–MS/MS experiment,the absolute abundance did not appear to play a role in generationof useful fragment ions for confident assignment of the glycopeptides
- In order to demonstrate that the detection of peptide backbone productions was a universal phenomenon, we analyzed glycopeptides with differentpeptide backbones and varying absolute abundances from each of thethree glycoproteins studied
- Table 2 summarizesthe types of product ions observed using HILC-C18 vs C18 LC–tandemMS
- Mass tables with observed and calculated fragment ion masses areshown in Supporting Information Section S-4
- For all glycopeptide assignments that were confirmed by tandemMS, the following criteria were used: (1) intact glycopeptide massmatch, (2) presence of oxonium ions, (3) presence of either an abundantprotonated peptide ion and/or a peptide + HexNAc ion, and (4) presenceof significant peptide backbone product ion coverage
- It is significantthat some of the glycopeptide com positions that were assigned to high-abundance precursor ions based on intact mass that also produced oxonium ionswere rejected (shown in red font) because the tandem MS stub glycopeptideor peptide backbone ions were not consistent with the assignment
- This emphasizes the need for tandem MS on glycopeptides for confidentassignments
- Although the ions shown in Table 2 were quite abundant in the mixtures analyzed, many were not selectedfor data-dependent tandem MS when using C18-MS due to the presenceof more abundant nonglycosylated peptides
- In addition, GlycReSoftfailed to find and match some of these com positions in C18 data, whichwas probably due to very low abundances or presence of overlappingisotopic peaks
- HILIC-C18-MS improved the abundances of glycopeptidesrelative to nonglycosylated peptides , which allowed these ions tobe matched by the GlycReSoft software
- Data-dependent acquisitionof tandem mass spectra led to systematic identification of these glycopeptidesand allowed confident assignments due to the presence of peptide backboneproduct ions
- The abundant product ions containing stub glycopeptidesand oxonium ions were useful for confirming peptide identities
Output (sent_index, trigger,
protein,
sugar,
site):
- 10. glycopeptide, , -, -, glycopeptide
- 11. glycopeptide, , -, -, glycopeptide
- 11. glycopeptides, , -, -, glycopeptides
- 13. glycosylation, , -, -, site
- 14. glycopeptide, , -, -, glycopeptide
- 14. glycopeptides, , -, -, glycopeptides
- 15. glycopeptides, , -, -, glycopeptides
- 15. glycoproteins, , glycoproteins, -, -
- 18. glycopeptide, , -, -, glycopeptide
- 19. glycopeptide, , -, -, glycopeptide
- 2. glycopeptide, , -, -, glycopeptide
- 20. glycopeptides, , -, -, glycopeptides
- 23. nonglycosylated, , -, -, peptides
- 4. glycopeptides, , -, -, glycopeptides
- 5. glycopeptide, , -, -, glycopeptide
- 6. glycopeptide, , -, -, glycopeptide
- 9. glycopeptide, , -, -, glycopeptide
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):