A synthetic camel anti-lysozyme peptide antibody (peptibody) with flexible loop structure identified by high-resolution affinity mass spectrometry.
We describe the synthesis and characterisation of the fully functional molecular recognition structure of a 26-amino acid residue peptide antibody, referred to as peptibody, designed from a monoclonal single-domain antibody fragment derived from a camel heavy-chain antibody. The CDR3 region (CDR = complementarity determining region) of the cAbLys3 camel antibody fragment, which binds to the active site of hen eggwhite lysozyme (HEL) and acts as a potent enzyme inhibitor by mimicking an oligosaccharide substrate, was prepared by solid-phase peptide synthesis. To obtain a closed loop-like structure resembling that in the crystal structure, N- and C-terminal cysteine residues were added to the linear peptide and oxidised to a cyclic disulfide-bridged peptide by using dimethylsulfoxide. A further, internal cysteine-12 residue was acetamidomethyl-protected to prevent possible oxidative byproducts. Affinity separation on a lysozyme microcolumn combined with MALDI-TOF mass spectrometry revealed that the peptide resumed high affinity to lysozyme only after deprotection of Cys-12, suggesting the importance of this paratope sequence for epitope recognition. The complex of lysozyme and active peptibody was characterised directly by conducting high-resolution ESI-FTICR mass spectrometry, which provided a molecular comparison of affinities for linear and cyclic peptibodies.
Marquardt
Andreas
A
Department of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, 78457 Konstanz (Germany).
Muyldermans
Serge
S
Przybylski
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000737:chemistry* / Q000494:pharmacology*; D002162:Camelus / Q000276:immunology*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003500:Cyclization; D003545:Cysteine / Q000737:chemistry; D016014:Linear Models; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000037:antagonists & inhibitors*; D010447:Peptide Hydrolases / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019138:Antibodies, Blocking; D009113:Muramidase; D010447:Peptide Hydrolases; D003545:Cysteine