| Abstract:
High site density photopolymer substrates have been constructed using scanning laser and micromirror array (MMA) systems to form ~250 µm diameter porous gel and hydrogel microstructures. Based on the absorbance of the dibenzofulvene-piperidine these structures have a reactive site density of ~50nmoles/feature. This high site density provides sufficient material for direct characterization of individual microstructures using common analytical techniques such as absorbance, fluorescence, and MALDI-MS. These microstructures are used as substrates for the construction of heteropolymer arrays using light directed and spotting methods. In the case of light directed methods, the addition of the photolabile protective group 6-nitroveratryloxycarbonyl (NVOC) results in increased swelling of the microstructures. This observation led to the construction of polymer microstructures that rapidly move upon asymmetric illumination with a laser beam. Peptide microarrays are constructed directly on the microstructures using the MMA NVOC after derivatization of the microstructures with an acid labile linker. Microstructures are also grafted with dsDNA containing three repeats of the consensus sequence for Human AP1 and NFkB, incubated with a solution of AP1, washed, and digested in situ with trypsin. Microarrays are characterized in situ using MALDI-MS after treating microstructures individually with a TFA and alpha-cyano-4-hydroxycinnamic acid solution. In situ peptide mass fingerprinting of microstructures with AP1 consensus sequence successfully identifies human AP1 in treatment areas and not in the control. The peptide GGFL-amide is detected as a sodium adduct (414.215 Da) and N-terminal labeling with N-Tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) is used to sequence the peptide TMPP-GGFL using post source decay analysis. This demonstrates the in situ identification of a DNA binding protein, identification, and sequencing of peptides using arrays of high site density polymer microstructures. Current work is focused on developing this technique to construct larger peptide arrays and using DNA arrays to study transcription factors in cell extracts including: NFKB, SP1, AP1, and GATA. Ultimately this work could result in a high throughput method to screen for various biomarkers.
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