On-chip analysis of protein-protein interaction using MALDI-TOF MS

Abstract

We demonstrated the strategy of selection and identification of analyte proteins of interest by combining a chip-based biosensor with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For this, the affinity surface of a biosensor chip was constructed over a dendrimer monolayer on gold by employing the interaction between bovine serum albumin (BSA) and its antibody. After trypsin digestion of captured BSA under optimized conditions, digested product was collected and analyzed through peptide mass fingerprinting with MALDI-TOF MS. The detection limit in this approach reached to about 100 femtomole level. The structural alignment of peptides obtained from on-chip digestion of 1pmol BSA revealed that exposed regions of BSA molecule, affinity-captured on an antibody layer, were likely to be trypsin-digested. Efficacy of a used chip platform to on-chip digestion was also tested in other antibody layers. In real applications of treating a protein mixture and human serum onto the prepared antibody layer, BSA can be detected down to a level of 0.1 ng/mL. Furthermore, specific proteins expressed in E.coli were successfully identified from a cell lysate. These results represent that combined use of a chip-based technique and MALDI-TOF MS is capable of identifying low concentration of analyte proteins in complex biological mixture.