Light-directed synthesis and in situ MALDI-MS characterization of complex bioheteropolymer microarrays

Light was used to construct and perform synthesis on arrays of porous crosslinked methacrylate polymer microstructures. The resulting DNA or peptide grafted microstructures represent complex bioheteropolymeric materials which were used to selectively bind and identify proteins using mass spectrometry. Light was modulated using scanning laser and micromirror array systems. Laser based photo polymerization results in the formation of pores without the use of porogens in regions of highest light flux. Porous structures were also achieved using the micromirror array in combination with porogenic solvents. Microstructures were aminated with Fmoc-amino acids and the absorbance of the dibenzofulvene-piperidine revealed site densities of amines of ∼0.1-1 nmole/feature. Photolabile blocking groups ((alpha-methyl-2-nitropiperonyl)oxy)carbonyl (MeNPOC) or 6-nitroveratryloxycarbonyl (NVOC) were used to modulate the reactivity of the N-terminal amine with light. Light directed patterning revealed the accumulation of colored and fluorescent photo-generated side products. These side reactions were inhibited by adding excess thiol compounds during photolysis. Photochemical cleavage of NVOC-Glycine grafted microstructures using asymmetric illumination with a 365nm laser induced bulk movement of the microstructure with a maximum velocity of 1 mm/s, presumably due to asymmetric changes in swelling. Light directed peptide synthesis on polymer microstructures containing an acid labile linker was performed using in situ substitution of McNPOC for N-terminal Fmoc groups and bromophenol blue staining to monitor stepwise yield. The resulting peptide arrays were individually treated with TFA, alpha-cyano-4-hydroxycinnamic acid, and characterized in situ using Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). MALDI-MS confirmed that the designed peptides had been synthesized on their respective array elements. Post source decay was used to sequence peptides cleaved from the polymer microstructures. In another case, DNA oligonucleotides containing the consensus sequence for transcription factor AP-1 (c-JUN) were immobilized on polymer structures activated with N,N'-Disuccinimidyl carbonate and annealed to a fluorescently labeled complimentary strand resulting in intense fluroescence that can be seen by eye. These were exposed to solutions containing rhAP-1, washed, and individually spotted with Trypsin, and then matrix. !n situ MALDI-MS revealed peptide fragments corresponding to rhAP-1. This represents a new and general method for constructing and characterizing complex bioheteropolymers arrays which may have broad proteomics applications.