Identification of Protein Interactions via Mass Spectroscopy
Produce Safety and Microbiology Research
2012 Annual Report
1a.Objectives (from AD-416):
The ultimate goal of this project is to identify proteins of interest in certain diseases chosen for study by Prosetta and ARS, and any locations of protein-protein and protein-ligand binding relevant to the mechanisms of those diseases, for use inter alia in understanding a mechanism of action (MOA) between the protein and a particular ligand to develop new diagnostics, therapeutics, and prophylactics.
1b.Approach (from AD-416):
Prosetta will provide ARS with putative protein targets that Prosetta will purify using a variety of affinity techniques; the proteins may include various modifications and cross-links. Final purification will be done by SDS-PAGE gels that will be stained with coomassie blue. The proteins of interest will be cut from the gels and then processed to yield a set of peptides, which will be analyzed by mass spectrometry to identify the sequences and structural features of the proteins. Further analysis will be performed to reveal the location of any modifications or crosslinks.
In FY12 ARS Scientists at Albany, California, used mass spectrometry to analyze a large number of samples collaborating with the cooperator (Prosetta). A number of proteins were identified as a result of this research collaboration. While the proteins are important to Prosetta, ARS is more interested in the methods developed for isolation and identification of interacting proteins. ARS is using this approach (among others) to identify partners for misfolding prion proteins and these partner molecules will be evaluated as potential surrogate markers for transmissible spongiform encephalopathy (TSE) diseases. This research relates to the objective of developing tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments.