title: Probing orientation of immobilized humanized anti-lysozyme variable fragment by time-of-flight secondary-ion mass spectrometry.
abstract: As methods to orient proteins are conceived, techniques must also be developed that provide an accurate characterization of immobilized protein orientation. In this study, X-ray photoelectron spectroscopy (XPS), surface plasmon resonance (SPR), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to probe the orientation of a surface immobilized variant of the humanized anti-lysozyme variable fragment (HuLys Fv, 26 kDa). This protein contained both a hexahistidine tag and a cysteine residue, introduced at opposite ends of the HuLys Fv, for immobilization onto nitrilotriacetic acid (NTA) and maleimide oligo(ethylene glycol) (MEG)-terminated substrates, respectively. The thiol group on the cysteine residue selectively binds to the MEG groups, while the his-tag selectively binds to the Ni-loaded NTA groups. XPS was used to monitor protein coverage on both surfaces by following the change in the nitrogen atomic %. SPR results showed a 10-fold difference in lysozyme binding between the two different HuLys Fv orientations. The ToF-SIMS data provided a clear differentiation between the two samples due to the intensity differences of secondary ions originating from asymmetrically located amino acids in HuLys Fv (histidine: 81, 82, and 110 m/z; phenylalanine: 120 and 131 m/z). An intensity ratio of the secondary ion peaks from the histidine and phenylalanine residues at either end of the protein was then calculated directly from the ToF-SIMS data. The 45% change in this ratio, observed between the NTA and MEG substrates with similar HuLys Fv surface coverages, indicates that the HuLys Fv fragment has opposite orientations on two different surfaces.
authors.affiliation: Department of Chemical Engineering, University of Washington, Box 351750, Seattle, Washington 98195-1750.
authors.forename: J E
authors.initials: JE
authors.lastname: Baio
authors.forename: Fang
authors.initials: F
authors.lastname: Cheng
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Ratner
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
authors.forename: David G
authors.initials: DG
authors.lastname: Castner
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000378:metabolism; D009113:Muramidase / Q000276:immunology*; D056951:Photoelectron Spectroscopy; D017434:Protein Structure, Tertiary; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods*; D020349:Surface Plasmon Resonance
chemical_list: D000596:Amino Acids; D055767:Immobilized Proteins; D007135:Immunoglobulin Variable Region; D009113:Muramidase