From SPRI (Surface Plasmon Resonance Imaging) Affinity Capture Analysis Up to On-Chip MALDI-MS/MS Analyte Identification

Abstract

Multiplex format SPRi analysis allows direct visualization and thermodynamic analysis of biomolecular interactions, and is advantageously used for ligand-fishing of captured bio-molecules on multiple immobilized receptors. Mass spectrometry is a powerful tool for structural characterization and identification. Therefore, the combination of SPRi and MS into one concerted procedure is of a great interest for functional and structural analysis in the fields of proteomics, drug-discovery or diagnostic. We have implemented an on-chip MALDI analysis in which affinity captured bio-molecules are directly detected from the SPR-sensor surface.The model presented was based on antigens-antibodies interactions. Antibodies, Anti-Beta-lactoglobulin, Anti-ovalbumin and a reference, were arrayed on biochip functionalized with a patented SAM-NHS surface chemistry and the SPR experiments were performed using a MS buffer. A mixture of label free antigens, Beta-lactoglobulin and ovalbumin, was injected and analyte capture was followed in real time. Following the collection of kinetic constants (Kon and Koff) information, the biochip was removed and each spot was submitted to in situ digestion treatment and MALDI matrix deposition. The SPRi sensor surface was inserted directly in an appropriate MALDI plate holder and MALDI-MS and MS/MS complete identification of the retained antigens was performed directly from each individual spots on the biochip. Using this process, the transfer of the biochip into the MALDI apparatus consecutive to a SPR imaging experiment was straightforward without intermediate treatment that could lead to sample loss and/or contaminations.A same surface array is dedicated to SPRi for affinity separation and subsequent MALDI-MS and MS/MS identification of the retained ligands from complex solution.