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Methods for multiplexed protein measurements

Biological systems are highly complex and dynamic, and their behavior is very difficult to predict only from knowledge of the individual parts. Any individual changes that irreversibly distort the flow of information in the network could result in pathological conditions and hence give rise to diseases such as cancer. To characterize signaling networks as a system, however, we first need technologies that allow us to rapidly and accurately quantify the abundances and post-translational modification states of many different proteins simultaneously. Working with Dr. Gavin MacBeath, I tailored the protein microarray technology for studying signaling networks in clinical samples.  

The following research studies illustrate various applications of protein microarray technology, including the investigation of protein-protein interactions and signaling networks in clinical samples.

 
Profiling phospho-signaling networks in tumor biopsies using lysate microarrays
(Reverse Phase Protein Arrays)

My research aims to deepen our understanding of the mechanisms that regulate cancer as well as design a method to predict and provide cancer treatment on an individual basis. The biggest problem in cancer treatment is that one kind of therapy does not work for all. Every patient arrives in the clinic at a different stage and has to be treated according to the stage or progression of cancer. I developed a general strategy to validate lysate microarray technology to accurately and reproducibly quantify the abundances and modification states of multiple proteins in very small clinical specimens. I asked whether array technology can be used to study signaling by receptor tyrosine kinases in extracts prepared from matched normal and cancer tissue from 56 breast patients. My analysis of the breast cancer signaling data shows that I can use this precise characterization of the activity status of several proteins to build “networks” and that each patient's tumor sample involves a distinct “topology”. Network maps can also serve to identify potentially new interactions. Ultimately, this approach has the potential to provide a means to map the connectivity of poorly understood signaling networks, and my findings may ultimately guide efforts to develop individualized therapies for cancer.

 Relevant Publications:
Gujral et al., 2012 (Oncogene)
Luckert & Gujral et al., 2012 (Science Signaling)
Gujral & MacBeath, 2010 (Science Signaling)
 
Discovery of protein-protein interactions using protein microarrays
The following studies show that biochemical interactions identified using protein microarrays in vitro can be used to uncover new biology, suggesting that constructing microarrays of protein interaction domains provides a viable way to segment the problem of identifying biologically meaningful protein-protein interactions on a proteome-wide scale.
PDZ domain-β-catenin interactions: I used a systems approach to discover novel PDZ domain-mediated interactions with β-catenin. Using protein microarrays comprising 206 mouse PDZ domains, we identified 26 PDZ domain-mediated interactions with β-catenin and subsequently confirmed them both in vitro and in cellular lysates. Many of the newly discovered interactions involved proteins with annotated roles in the formation or maintenance of tight junctions such as Scrib, Magi-1, Pard3, and ZO-3. Disrupting these interactions affects cellular adhesion, proliferation, and migration, consistent with a model in which β-catenin plays an integral role in maintaining cell–cell contacts, participating not only in adherens junctions, but also in tight junctions.
PDZ-PDZ dimerization: PDZ domains are best known for mediating protein-protein interactions by binding the C-termini of their target proteins. They have also been reported, however, to dimerize with other PDZ domains. To further characterize this alternative binding mode, we undertook an unbiased, proteome-wide investigation of mammalian PDZ-PDZ interactions using protein microarrays, fluorescence polarization, and co-affinity purification. In total, we identified 37 PDZ-PDZ interactions involving ~30% of all PDZ domains tested, indicating that PDZ-PDZ dimerization occurs at a higher frequency than previously appreciated. This suggests that many PDZ domains evolved to form multiprotein complexes by simultaneously interacting with more than one ligand.
 
Relevant Publications:
Gujral & Karp et al., 2013 (Chem & Biol.)

Chang & Gujral et al., 2011 (Chem & Biol.)