Optimizing microarray-based in situ transcription–translation of proteins for matrix-assisted laser desorption ionization mass spectrometry

The analysis of self-assembled protein microarrays, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, combines two high-throughput platforms for investigation of the proteome. In this article, we describe the fabrication in situ of protein arrays optimized for MALDI characterization. Using the green fluorescent protein (GFP) both as an epitope for immobilization and as a gauge for relative protein expression, we were able to generate amounts of protein on the array slides sufficient for MALDI identification. In addition, expression of N-terminal protein constructs fused to GFP demonstrated mass shifts consistent with that of the full-length protein. We envision this technology to be important for the functional screening of protein interactions.