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

Abstract

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 report, 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 also 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.

Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis

“…According to the slide manufacturers, due to the hydrophilic nature of the coating, the spot size would be slightly larger than that achieved with coatings that are more hydrophobic 44 . Kimzey and co-authors, after printing GFP fluorescence protein on various chemistry surfaces from different manufacturers, reported that N-hydroxysuccinimide (NHS)-ester coated slides were able to bind significantly higher amount of protein compared to epoxide and aldehyde coated surfaces 45 . Yang et al .…”

“…| (2020) 10:18208 | https://doi.org/10.1038/s41598-020-75226-y www.nature.com/scientificreports/ were able to bind significantly higher amount of protein compared to epoxide and aldehyde coated surfaces 45 . Yang et al reported similar findings, after examining the relationship between the cell number and the surface chemistries analysed by ToF-SIMS 46 .…”

Solid-Phase Biological Assays for Drug Discovery

“…The miniaturized format provides a method for increased sample throughput and multiplexed analyte detection, and requires smaller reaction volumes, which allows for increased sample concentrations and reaction kinetics versus plate-based assays (9). In addition, microarrays can be printed into 96 well plates to allow integration with conventional liquid handling systems and can be imaged by a range of techniques, including colorimetry (69), fluorescence (70,71), SPR (72,73), and mass spectrometry (MS) (74,75), providing the ability to design a wide range of assays to study protein-ligand interactions.…”