Elucidating Novel Protein Interactions Using MALDI-MS and a Self-Assembled Monolayer Chip
Summary The ability for real-time identification of protein binding from whole cell lysates is a challenge for protein microarrays. This invention uses MALDI-MS to analyze complex biological mixtures using a protein or small molecule microarray, which is characterized by a self-assembled monolayer on gold contacts. Specialized surface chemistry enables efficient creation of both small molecule and protein microarray chips. For protein attachment to the chip, self-assembled monolayers of alkanethiolates on gold are utilized, in addition to an oligo(ethylene glycol) chain that resists adsorption of proteins. For immobilization of small molecules to the chip, a surface meleimide has been synthesized.
Whole cell lysates are incubated with microarray targets, followed by an in-situ proteolytic digestion step (without a subsequent washing/desalting or chromatography step), and MALDI-MS analysis. Proof-of-concept studies unambiguously identified proteins from a crude HeLa cell lysate, suggesting a highly sensitivity, high throughput protein analysis tool.
Applications The need for high throughput protein analysis will address applications in clinical diagnostics and protein/small molecule screening, which may include:
-Protein analysis of crude samples as a clinical diagnostic -Natural product identification to a specific protein -Analysis of unknown proteins (e.g., whole cell lysate) with other unknown proteins immobilized to the chip (e.g., individual proteins form an unknown cDNA library)
-Use for enzymatic assays (e.g., phosphorylations, dephosphorylations, glycosylation, proteolysis)
-Extension of technology to quantitative methods, e.g., ICAT, AQUA, etcâ€¦ For Further Information Please Contact the Director of Business Development Michal Preminger Email: firstname.lastname@example.org Telephone: (617) 432-0920
Kirschner, Marc W.
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