The HARPER Lab

Department of Cell Biology

Quantitative Proteomics of Signaling Networks

Quantitative analysis of CRL complexes by AQUA proteomics

Proteome networks undergo reorganization in response to cellular signaling. This often results in either protein turnover, or changes in the modification state of proteins. However, understanding how networks are reorganized in response to perturbations such as small molecule inhibitors of a pathway are often difficult, and require quantitative approaches. We are applying multiple quantitative proteomic approaches to discern how various signaling networks are altered in response to pathway perturbation. We have performed a systematic analysis of the cullin-RING E3 ligase network in response to inhibition of the neddylation system using a small molecule inhibitor. This work has revealed that the abundance of cullin adaptor proteins in the cell control the assembly state of the CRL network, and there is little evidence for the involvement of CAND1 in global sequestration of cullins in response to acute dennedylation. These studies employ multiplex AQUA (absolute quantification) as a tool to survey the concentrations for more than 14 proteins in the CRL network simultaneously. We are currently developing this approach further with the intent to characterize dynamic changes in tumor suppressor and oncogenic signaling systems.