AVS 56th International Symposium & Exhibition
    Biomaterial Interfaces Thursday Sessions
       Session BI-ThP

Paper BI-ThP18
Probing Orientation of Immobilized Humanized Anti-Lysozyme Variable Fragment by Time-of-Flight Secondary Ion Mass Spectrometry

Thursday, November 12, 2009, 6:00 pm, Room Hall 3

Session: Biomaterial Interfaces Poster Session II (Arrays, Sensing, Micro/Nanofabrication, SPM)
Presenter: J.E. Baio, University of Washington
Authors: J.E. Baio, University of Washington
F. Chen, University of Washington
L.J. Gamble, University of Washington
D.G. Castner, University of Washington
Correspondent: Click to Email
The ability to orient proteins on surfaces to control exposure of their biologically active sites benefits a wide range of applications including protein microarrays, antibody-based diagnostics, affinity chromatography, and biomaterials that present ligands to bind cell receptors. As methods to orient proteins are developed, techniques must also be developed that provide an accurate characterization of immobilized protein orientation. In this study, x-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to probe the orientation of a surface immobilized variant of humanized anti-lysozyme variable fragment (HuLys Fv, 26kDa). This protein contained both a His-tag and a cysteine residue, introduced at opposite ends of the HuLys Fv. Previously, we have shown that we could successful control orientation of a Protein G fragment via a cysteine-maleimide bond. To induce opposite end-on orientations of the HuLys Fv variant, it was immobilized onto maleimide oligo(ethylene glycol) (MEG) and nitrilotriacetic acid (NTA) terminated substrates. The thiol group on the cysteine residue will selectively bind to the MEG groups, while the His-tag will selectively bind to the Ni loaded NTA groups. Protein coverage, on both surfaces, was monitored by the change in the atomic % of N, as observed by XPS. The height of the immobilized protein (3nm) was larger than the typical sampling depth of ToF-SIMS, consequently it only samples the top portion of the protein. This was confirmed by principal component analysis (PCA) of the ToF-SIMS results, which demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically in HuLys Fv (Histidine: 81, 82, and 110 m/z; Phenylalanine: 120 and 131 m/z). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of secondary ions stemming from the histidine and phenylalanine residues at either end of the protein. The three-fold increase in this ratio, observed between the MEG and NTA substrates, indicated opposite orientations of the HuLys Fv fragment on the two different surfaces.