title: Analysis of noncovalent chitinase-chito-oligosaccharide complexes by infrared-matrix assisted laser desorption ionization and nanoelectrospray ionization mass spectrometry.
abstract: Transferring noncovalently bound complexes from the condensed phase into the gas phase represents a challenging task due to weak intermolecular bonds that have to be maintained during the phase transition. Currently, electrospray ionization (ESI) is the standard mass spectrometric (MS) technique to analyze noncovalent complexes. Although infrared matrix-assisted laser desorption ionization (IR-MALDI)-MS also provides particular soft desorption/ionization conditions, this method has so far hardly been applied for the analysis of noncovalent complexes. In this study, we employed IR-MALDI orthogonal time-of-flight (o-TOF)-MS in combination with the liquid matrix glycerol to characterize the specific complex formation of chito-oligosaccharide (CHOS) ligands with two variants of Chitinase A (ChiA) from Serratia marcescens, the inactive E315Q mutant and the active W167A mutant, respectively. The IR-MALDI-o-TOF-MS results were compared to those obtained using nano-ESI-quadrupole (q)-TOF-MS and ultraviolet (UV)-MALDI-o-TOF-MS. Using IR-MALDI-o-TOF-MS, specific noncovalent complexes between ChiA and CHOS were detected with distributions between enzymes with bound oligosaccharides vs free enzymes that were essentially identical to those obtained by nano-ESI-q-TOF-MS. Chitinase-CHOS complexes were not detected when UV-MALDI was employed for desorption/ionization. The results show that IR-MALDI-MS can be a valuable tool for fast and simple screening of noncovalent enzyme-ligand interactions.
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælandsvei 6-8, N-7491 Trondheim, Norway.
authors.forename: Anette I
authors.initials: AI
authors.lastname: Dybvik
authors.forename: Anne Line
authors.initials: AL
authors.lastname: Norberg
authors.forename: Veronika
authors.initials: V
authors.lastname: Schute
authors.forename: Jens
authors.initials: J
authors.lastname: Soltwisch
authors.forename: Jasna
authors.initials: J
authors.lastname: Peter-Katalinić
authors.forename: Kjell M
authors.initials: KM
authors.lastname: Vårum
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.forename: Klaus
authors.initials: K
authors.lastname: Dreisewerd
authors.forename: Michael
authors.initials: M
authors.lastname: Mormann
authors.forename: Morten
authors.initials: M
authors.lastname: Sørlie
mesh_terms: D019943:Amino Acid Substitution; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics; D007259:Infrared Rays; D009154:Mutation; D036103:Nanotechnology / Q000379:methods; D009844:Oligosaccharides / Q000737:chemistry*; D012706:Serratia marcescens / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D009844:Oligosaccharides; D002688:Chitinases