AVS 50th International Symposium
    Biomaterial Interfaces Monday Sessions
       Session BI-MoM

Paper BI-MoM1
Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Surface Plasmon Resonance (SPR) Determination of Surface Bound Anti-Lysozyme Orientation

Monday, November 3, 2003, 8:20 am, Room 307

Session: Protein-Surface Interactions
Presenter: N. Xia, University of Washington
Authors: N. Xia, University of Washington
N.T. Samuel, University of Washington
P.S. Stayton, University of Washington
D.G. Castner, University of Washington
Correspondent: Click to Email
Static time-of-flight secondary ion mass spectroscopy (ToF-SIMS) and surface plasmon resonance (SPR) were used to analyze the orientation of immobilized proteins. A model protein with a well-define structure, the Fv fragment of a humanized anti-lysozyme (HuLys), was used. A His-tag linked to the C-terminal of the heavy chain was located opposite to the lysozyme binding domain (complementarity-determining region, CDR). We immobilized nitrilotriacetic acid (NTA) onto a self-assembled monolayer (SAM) of oligo(ethylene glycol)-terminated (OEG) thiol on Au. OEG provides a low-fouling background and NTA binds nickel ions, leaving two coordination sites available for interaction with the His-tag of HuLys. SPR experiments showed after nickel activation the NTA/OEG surface specifically bound HuLys Fv via the His-tag and that the immobilized HuLys Fv had nearly full antigen (lysozyme) binding capacity, suggesting a uniform CDR-exposed orientation of HuLys Fv on the surface. For comparison, we also activated the OEG SAM with carbonyldiimidazole (CDI), which binds protein via amine/imidazolyl carbamate chemistry and should result in a random protein orientation. This was supported by the SPR results, which showed that only ~50% of HuLys Fv immobilized on the CDI/OEG surface had antigen binding capability. The ToF-SIMS spectra of HuLys Fv immobilized on NTA/OEG and CDI/OEG surfaces were then compared. It was found that the peaks at m/z=81, 82 and 110 had lower intensities in the spectra of HuLys Fv immobilized on the NTA/OEG surface. These peaks all correspond to the primary mass fragments from the amino acid histidine. Since wild-type Hulys Fv has only 2 histidine residues, the ToF-SIMS results confirmed that HuLys Fv was immobilized onto the NTA/OEG substrate via the His-tag, which should be located at the bottom of the protein layer.