IDBR: TYPE A Development of On-Chip Electrochemical Cross-Linking Mass Spectrometry for Probing Protein-Protein Interactions

  • Wu, Shiyong S. (PI)
  • Chen, Hao (CoPI)
  • Wu, Shiyong S. (CoPI)

Project: Research project

Project Details

Description

An Award is made to Ohio University to develop an on-chip electrochemical cross-linking mass spectrometry which will be used to identify protein-protein interactions (PPIs) in vivo. The study of PPIs is very important to understand protein biological functions as the biological functions of almost all proteins are provided by specific, non-covalent interactions with other molecules. The identification of interacting partners and interaction sites of protein complexes or aggregates will shed light on the elucidation of biological process mechanisms. This instrumentation project will provide opportunities for students to be trained in interdisciplinary research involving analytical chemistry, biochemistry, and instrumentation. The proposed instrument for biological research will first be used for the study of UV-induced apoptotic signaling pathways. Following instrument construction and software training, many other biological research groups in structural and molecular biology at Ohio University will have access to the developed instrument. The instrument will also serve as a prototype instrument for commercialization, enabling dissemination to the biological research community. It is expected that the proposed research will benefit the research communities of structural biology, molecular biology, and drug discovery.

Cross-linking mass spectrometry (MS) has become a useful technique for mapping protein-protein interactions and elucidating protein networks in living cells. However, several experimental obstacles limit the usefulness of cross-linking MS, including difficulty in the identification of cross-linked peptides in the complex mixture, the complexity of the fragmentation patterns of cross-linked peptide ions and the inability to quantify cross-links. This project proposes a new approach using electrochemistry (EC)-assisted cross-linking MS for probing protein-protein interactions in vivo, based on an electrochemical cross-linking reagent, diselenide [succinimidyl propionate] (SSP). Following online digestion, the cross-linked protein complexes will undergo capillary electrolysis (CE) separation, online EC reduction and online MS detection. The EC reduction provides a novel way to quickly identify and quantify cross-links based on the reduction current (also on MS signal), significantly reducing time required for data analysis by more than one order of magnitude. The online EC reduction takes seconds, which is much faster than hours required in chemical reduction. Furthermore, EC reduction yields easily identifiable linear peptides, facilitating MS/MS identification of cross-link structures for pinpointing out the interaction sites in the protein complexes.

StatusFinished
Effective start/end date4/15/153/31/19

Funding

  • National Science Foundation: $322,727.00

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