Peptide and small molecule microarray for high throughput cell adhesion and functional assays.
A novel class of chemical microchips consisting of glass microscope slides was prepared for the covalent attachment of small molecule ligands and peptides through site-specific oxime bond or thiazolidine ring ligation reaction. Commercially available microscope slides were thoroughly cleaned and derivatized with (3-aminopropyl)triethoxysilane (APTES). The amino slides were then converted to glyoxylyl derivatives via two different routes: (1) coupling of Fmoc-Ser followed by deprotection and oxidation, or (2) coupling with protected glyoxylic acid and final deprotection with HCl. Biotin or peptide ligands derivatized at the carboxyl terminus with a 4,7,10-trioxa-1,13-tridecanediamine succinimic acid linker and an amino-oxy group or a 1,2-amino-thiol group (e.g., cysteine with a free N(alpha)-amino group) were printed onto these slides using a DNA microarray spotter. After chemical ligation, the microarray of immobilized ligands was analyzed with three different biological assays: (1) protein-binding assay with fluorescence detection, (2) functional phosphorylation assay using [gamma(33)P]-ATP and specific protein kinase to label peptide substrate spots, and (3) adhesion assay with intact cells. In the cell adhesion assay, not only can we determine the binding specificity of the peptide against different cell lines, we can also determine functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip. This chemical microchip system enables us to rapidly analyze the functional properties of numerous ligands that we have identified from the "one-bead one-compound" combinatorial library method.
Falsey
J R
JR
UC Davis Cancer Center, Division of Hematology/Oncology, and Department of Internal Medicine, University of California Davis, 4501 X Street, Sacramento, California 95817, USA.
Renil
M
M
Park
S
S
Li
S
S
Lam
K S
KS
D001345:Autoradiography; D002448:Cell Adhesion; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D006801:Humans; D008024:Ligands; D008832:Microchemistry; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000138:chemical synthesis; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011485:Protein Binding; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D008024:Ligands; D009842:Oligopeptides; D010761:Phosphorus Radioisotopes