IUVSTA 15th International Vacuum Congress (IVC-15), AVS 48th International Symposium (AVS-48), 11th International Conference on Solid Surfaces (ICSS-11)
    Biomaterials Thursday Sessions
       Session BI-ThP

Paper BI-ThP5
Time-of-flight Secondary Ion Mass Spectrometry (ToF-SIMS) Characterization of Adsorbed Proteins with Amino Acid Mutations

Thursday, November 1, 2001, 5:30 pm, Room 134/135

Session: Biomolecule and Cell Poster Session
Presenter: N. Xia, University of Washington
Authors: N. Xia, University of Washington
R. To, University of Washington
S.L. McArthur, 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 spectrometry (ToF-SIMS) was used to detect amino acid sequence mutations in adsorbed streptavidin. A series of mutations were introduced into wild-type streptavidin using site-directed mutagenesis. Both wild-type and mutant strepavidin were adsorbed onto polystyrene. Then positive and negative static ToF-SIMS spectra were acquired. For mutants where one amino acid (either glutamic acid or serine) in each strepavidin monomer was replaced with a cysteine the largest differences were observed in the negative spectra. The appearance of ToF-SIMS peaks at 32 (S@super -@) and 33 (HS @super -@) indicated the incorporation of cysteine into the streptavidin mutants. Only small changes in the positive ion ToF-SIMS spectra were observed upon incorporation of cysteine into streptavidin (e.g., the appearance of a small peak at 45 due to the CHS @super +@ fragment from cysteine). Introduction of a larger mutation (linking a histidine tag to the C-terminus of each streptavidin monomer) resulted in larger changes in the positive ion ToF-SIMS spectra. Due to the relative complexity of the positive spectra from proteins, principal component analysis (PCA) was used to analyze the differences in the positive spectra. The largest changes introduced by the presence of the histidine tags were observed in the intensities of the peaks at m/z=81, 82 and 110, which correspond to the primary mass fragments from the amino acid histidine.