AVS 56th International Symposium & Exhibition
    Biomaterial Interfaces Tuesday Sessions
       Session BI-TuM

Paper BI-TuM5
Investigation of Surface-Bound Protein Conformation/Orientation Using Time-of-Flight Secondary Ion Mass Spectrometry

Tuesday, November 10, 2009, 9:20 am, Room K

Session: Proteins and Cell Interactions at Interfaces II
Presenter: L.J. Gamble, University of Washington
Authors: L.J. Gamble, University of Washington
F. Cheng, University of Washington
J. Brison, University of Washington
L. Árnadóttir, University of Washington
D.G. Castner, University of Washington
Correspondent: Click to Email
The adsorption of protein to solid surfaces is typically accompanied by structural rearrangements as well as loss of bioactivity. These changes can be monitored by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the protein activity monitored by surface plasmon resonance (SPR). However, the ultra-high vacuum of the ToF-SIMS can alter the protein conformation. In this study ToF-SIMS was coupled with a variable temperature sample stage to monitor the conformational changes that occur when a surface-bound protein goes from a hydrated to a dehydrated state. Changes in bioactivity of the surface bound proteins were investigated using SPR. Initial ToF-SIMS and SPR experiments were conducted on a surface-bound protein system of histagged humanized anti-lysozyme variable fragment (HuLys Fv) coordinated on a Ni2+-loaded nitrilotriacetic acid (NTA) surface. Continuing studies investigate fibrinogen (and fibrinogen fragments). Positive ToF-SIMS data from the protein surfaces were acquired with an ION-TOF TOF.SIMS 5-100 system (ION-TOF GmbH, Münster, Germany) . Applying principal component analysis (PCA) to the ToF-SIMS data, the spectral differences resulting from two surface coverages and various heat treatments were determined. The spectra are separated into three groups: high protein coverage samples, low coverage below -80oC, and low coverage at -60oC and above. Trends observed in the plot suggest both surface coverage and heat treatment affected the secondary ion spectra. At the temperature below about -80oC, the protein molecules are frozen into their hydrated conformation. As the temperature is raised changes expose hydrophobic amino acid residues. The antigen binding capacity of surface-bound HuLys Fv before and after dehydration was measured by SPR. At the low coverage, the antigen binding capacity on the dried protein film was roughly 50% lower than that on the fresh film. As comparison, high coverage dried samples lost ~20% binding capacity. The loss of HuLys Fv bioactivity on the dried protein film was attributed to an irreversible disruption of protein native conformation during the drying process. The high coverage samples exhibited less loss of bioactivity, consistent with the smaller conformational changes observed by PCA. The use of the sugar trehalose as a protein stabilizer is also investigated.