Protein Cross-Linking Technology and Applications

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With support from the Chemical Measurement and Imaging program in the Division of Chemistry, and partial co-funding from the Cellular Dynamics and Function program in the Division of Molecular and Cellular Biosciences, Professor James Reilly and his group at Indiana University are developing experimental and computational methods to better characterize the complex interactions among proteins that are critical to the function (and misfunction) of biological cells. Their work addresses interactions such as protein folding and misfolding (implicated in conditions such as Alzheimer's or Parkinson's disease), and the chemistry of antibodies relevant to treating certain diseases. Graduate students, undergrads and high school students who participate in this research learn about the interplay of physical, analytical and biochemistry. They are also involved in the dissemination of research results through publications and conference presentations that prepare them for further scientific training or careers in academia, biotechnology and the pharmaceutical industry.Cross-linking mass spectrometry is a method of studying protein-protein interactions that is growing in popularity. The isolation and identification of protein cross-links is nevertheless still filled with challenges. Work in the Reilly laboratory aims to contribute to this field by improving the degree of confidence of protein cross-link identifications. Multiple fragmentation methods, highlighted by novel cross-linkers that are cleavable by electron transfer dissociation, are being utilized to increase the information content in mass spectral data. Data fusion seeking both qualitative and quantitative information is a significant challenge. Ion exchange and isoelectric focusing are used to enrich and distinguish linked proteins from unlinked counterparts. Protein dimerization thermodynamics, the temperature dependence of protein unfolding, and interactions between ribosomes and associated proteins are among potential applications testing the relative performance of thioimidoesters and commercially available succinimidyl ester cross-linkers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.