Surface analysis instrumentation provides significant information about the protein immobilization process and the structure of immobilized proteins. Our recent studies have used Protein G B1 variants to examine protein orientation when the size of the protein is similar to the ToF-SIMS sampling depth (~2-3nm). Two variants with a single cysteine introduced at either end were immobilized via the cysteine thiol onto maleimide oligo(ethylene glycol) (MEG) self-assembled monolayers (SAMs). XPS was used to measure the amount of immobilized protein and ToF-SIMS was used to measure the protein orientation and to confirm covalent attachment of protein. ToF-SIMS data showed enrichment of mass peaks from amino acids located at the end of the protein opposite the cysteine surface position, consistent with end-on protein orientations. ToF-SIMS also showed a charged mutant of Protein G B1 was immobilized in opposite orientations on positively (amine) and negatively (carboxylic acid) terminated SAMs.

ToF-SIMS characterization of immobilized Protein G orientation

243rd National Meeting (2012)

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