Surface plasmon resonance imaging studies of protein interactions onto peptide and protein microarrays

The interactions of proteins to peptide and protein probes immobilized in array formats were investigated using surface plasmon resonance (SPR) imaging. Two array fabrication methods were developed to create arrays of oriented peptides and proteins using a combination of chemically modified self-assembled monolayers and microfluidic patterning. These arrays were applied to study a variety of protein-protein interactions demonstrating the potential of this technique for proteomics research.; Peptide arrays were created to examine the sequence specificity of peptide-protein interactions. A two-step process was used to prepare peptide arrays: (i) a set of microchannels was used to deliver chemical reagents to covalently attach peptide probes to the surface by a thiol-disulfide exchange reaction; (ii) a second microchannel was used as a small-volume flow cell (5--10 mul) to deliver protein solutions to the peptide array surface. Specifically, these arrays were applied to study the interactions of anti-FLAG to the FLAG epitope tag. Differential anti-FLAG adsorption was observed to peptides differing from the FLAG binding motif by a single amino acid substitution. Binding constants were measured for anti-FLAG interactions to four peptides immobilized on one chip using Langmuir isotherms. Peptide arrays were also applied to study the kinetics of protein adsorption/desorption using real-time SPR imaging. Kinetic measurements of S protein to S peptide derivatives were analyzed to determine desorption rate constants (kd), adsorption rate constants, and equilibrium adsorption constant (KAds). In addition, real-time SPR imaging measurements of peptide arrays were applied to study the surface enzymatic activities of the protease factor Xa.; Histidine-tagged fusion protein arrays were developed using nitrilotriacetic acid (NTA) capture probes on gold thin films to study protein-protein and protein-DNA interactions. SPR imaging measurements were employed to characterize the immobilization and specificity of his-tagged fusion proteins to the NTA surface. SPR imaging measurements were also used with the his-tagged fusion protein arrays to study multiple antibody-antigen binding interactions, and to monitor the sequence specific interaction of double-stranded DNA with TATA box-binding protein.