Development of multiple genome-wide proteome microarrays comprised wafer substrate-based chip and its scanner: An...

Proteome microarray technology enables high-throughput analysis of protein interactions with all kinds of molecules. Wafer (6-inch) substrates offer a promising alternative to conventional glass (2.6 × 7.6 cm) substrates for carrying proteomes. This study aims to develop high-density wafer-based proteome microarrays and a corresponding fluorescence scanner. We constructed E. coli proteome microarrays and probed them with the antimicrobial peptide indolicidin to identify its protein targets, revealing its antimicrobial mechanisms. Compared to glass substrates, wafer substrates showed a detectable fluorescence signal of the immobilized Dylight 550-labeled antibody at a lower concentration (200 ng/mL vs. 5000 ng/mL), indicating greater sensitivity. Spot images on wafers also exhibited a more uniform circular profile. We fabricated a wafer holder compatible with a regular glass microarray printer and successfully printed six entire genome-wide E. coli proteome microarrays, totaling approximately 52,000 protein spots, on one wafer. Probing the wafer array with indolicidin and its control in triplicate, we identified 75 E. coli K12 protein targets, many of which are enriched in transport functions. Notably, we also found that two proteins crucial for DNA synthesis (nrdF and nrdB) were targeted by indolicidin. This explains the earlier finding that indolicidin inhibits DNA synthesis in E. coli. This study introduces the first wafer-based proteome microarrays, demonstrating enhanced sensitivity and the ability to perform simultaneous multiplexed probing compared to regular glass slide-based proteome microarrays.