Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Derek Bishop - Vassar College
Co-Author(s): Philip Andrews, University of Michigan, Ann Arbor, MI; Manolo Palencia, University of Michigan, Ann Arbor, MI
Crosslinking combined with mass spectrometry has the potential to both quantify and identify numerous protein interactions and conformations simultaneously. Perfected, this technique would alter how drug target sites are identified and allow new proteins/ complexes that are not compatible with X-ray crystallography or NMR to be studied. The Andrews lab has engineered a new crosslinker (DC4) to be cleavable within a mass spectrometer, resulting in smaller crosslinked peptides, and better identification. To study the effectiveness of DC4 coupled with mass spectrometry, we conducted a pilot study using calmodulin as a model protein. Calmodulin was crosslinked in light DC4 in conditions promoting the dumbbell conformation and in C13 DC4 in conditions promoting the collapsed state. These reactions were quenched, combined and digested. Mass spec analysis showed high DC4 coverage of calmodulin along with certain crosslinks only appearing in the heavy or light reactions. We observed a significantly higher abundance of light DC4 crosslinks that we hypothesized has to do with the unbalanced rates of digestion of the closed and open states of calmodulin.
Funder Acknowledgement(s): NIH ; IREU in the Structure and Function of Proteins at the university of Michigan
Faculty Advisor: Philip Andrews, firstname.lastname@example.org
Role: All cross linked sample preparation and mass spec data analysis. Dr. Andrews and Manolo Palencia provided insight and guidance.