title: ETD allows for native surface mapping of a 150 kDa noncovalent complex on a commercial Q-TWIMS-TOF instrument.
abstract: Top-down approaches for the characterization of intact proteins and macromolecular complexes are becoming increasingly popular, since they potentially simplify and speed up the assignment process. Here we demonstrate how, on a commercially available Q-TWIMS-TOF instrument, we performed top-down ETD of the native form of tetrameric alcohol dehydrogenase. We achieved good sequence coverage throughout the first 81 N-terminal amino acids of ADH, with the exception of a loop located on the inside of the protein. This is in agreement with the exposed parts of the natively folded protein according to the crystal structure. Choosing the right precursor charge state and applying supplemental activation were found to be key to obtaining a high ETD fragmentation efficiency. Finally, we briefly discuss opportunities to further increase the performance of ETD based on our results.
authors.affiliation: Biomolecular and Analytical Mass Spectrometry group, Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, 2020, Antwerpen, Belgium.
authors.forename: Frederik
authors.initials: F
authors.lastname: Lermyte
authors.forename: Albert
authors.initials: A
authors.lastname: Konijnenberg
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Williams
authors.forename: Jeffery M
authors.initials: JM
authors.lastname: Brown
authors.forename: Dirk
authors.initials: D
authors.lastname: Valkenborg
authors.forename: Frank
authors.initials: F
authors.lastname: Sobott
mesh_terms: D000426:Alcohol Dehydrogenase / Q000032:analysis / Q000737:chemistry; D000595:Amino Acid Sequence; D004867:Equipment Design; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*
chemical_list: D005656:Fungal Proteins; D021122:Protein Subunits; D000426:Alcohol Dehydrogenase