title: Top-down tandem mass spectrometry on RNase A and B using a Qh/FT-ICR hybrid mass spectrometer.
abstract: 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.
authors.affiliation: Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Bourgoin-Voillard
authors.forename: Nancy
authors.initials: N
authors.lastname: Leymarie
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Costello
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012259:Ribonuclease, Pancreatic / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*
chemical_list: D006023:Glycoproteins; D010446:Peptide Fragments; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic
keywords: Collision-induced dissociation (CID); Electron capture dissociation (ECD); Electron transfer dissociation (ETD); Glycoproteomics; Infrared multiphoton dissociation (IRMPD)