Journal
PROTEOMICS
Volume 14, Issue 10, Pages 1174-1184Publisher
WILEY-BLACKWELL
DOI: 10.1002/pmic.201300433
Keywords
Collision-induced dissociation (CID); Electron capture dissociation (ECD); Electron transfer dissociation (ETD); Glycoproteomics; Infrared multiphoton dissociation (IRMPD)
Funding
- NIH [P41 RR010888/GM104603, S10 RR025082]
- NHLBI [HHSN268201000031C]
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Protein characterization using top-down approaches emerged with advances in high-resolution mass spectrometers and increased diversity of available activation modes: collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD) electron capture dissociation (ECD), and electron transfer dissociation (ETD). Nevertheless, top-down approaches are still rarely used for glycoproteins. Hence, this work summarized the capacity of top-down approaches to improve sequence coverage and glycosylation site assignment on the glycoprotein Ribonuclease B (RNase B). The glycan effect on the protein fragmentation pattern was also investigated by comparing the fragmentation patterns of RNase B and its nonglycosylated analog RNase A. The experiments were performed on a Bruker 12-T Qh/FT-ICR SolariX mass spectrometer using vibrational (CID/IRMPD) and radical activation (ECD/ETD) with/without pre- or post-activation (IRMPD or CID, respectively). The several activation modes yielded complementary sequence information. The radical activation modes yielded the most extensive sequence coverage that was slightly improved after a CID predissociation activation event. The combination of the data made it possible to obtain 90% final sequence coverage for RNase A and 86% for RNase B. Vibrational and radical activation modes showed high retention of the complete glycan moiety (>98% for ETD and ECD) facilitating unambiguous assignment of the high-mannose glycosylation site. Moreover, the presence of the high-mannose glycan enhanced fragmentation around the glycosylation site.
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