On-line bioaffinity-electrospray mass spectrometry for simultaneous detection, identification, and quantification of protein-ligand interactions

Abstract

We describe here an on-line combination of a surface acoustic wave (SAW) biosensor with electrospray ionization mass spectrometry (SAW-ESI-MS) that enables the direct detection,identification, and quantification of affinity-bound ligands from a protein ligand complex on a biosensor chip. A trapping column was used between the SAW-biosensor and the electrospray mass spectrometer equipped with a micro-guard column, which provides simultaneous sample concentration and desalting for the mass spectrometric analysis of the dissociated ligand. First applications of the on-line SAW-ESI-MS combination include (1), differentiation of â-amyloid (Aâ) epitope peptides bound to anti-Aâ antibodies; (2), the identification of immobilized Substance P peptide calmodulin complex; (3), identification and quantification of the interaction of 3-nitrotyrosine-modified peptides with nitrotyrosine-specific antibodies; and (4), identification of immobilized anti-á-synuclein human á-synuclein complex. Quantitative determinations of protein ligand complexes by SAW yielded dissociation constants (KD) from micro-to low nanomolar sample concentrations. The on-line bioaffinity-ESI-MS combination presented here is expected to enable broad bioanalytical application to the simultaneous, label-free determination and quantification of biopolymer-ligand interactions, as diverse as antigen-antibody and lectin-carbohydrate complexes.