MultiPep Customer Highlight: Dr. Andy Hudmon
Andy Hudmon,an Associate Professor of Medicinal Chemistry and Molecular Pharmacology in the College of Pharmacy at Purdue University, sat down with CEM to detail his study of calcium-dependent regulation of ion channels and mechanisms underlying substrate selection and targeting by protein kinases. Dr. Hudmon’s laboratory owns a Liberty Blue microwave peptide synthesizer and a MultiPep robotic peptide synthesizer with access to a second MultiPep, shared within the department.
Q: How did your research interests and goals develop?A: It started with my MS in Physiology at Auburn University in the laboratory of Dr. Jim Sartin studying Growth Hormone regulation via estrogen. From there, I moved on to my PhD from the University of Texas Health Science Center at Houston, where I trained in the laboratory of Dr. Neal Waxham studying the structure/function of CaMKII in excitotoxic neurodegeneration. As a postdoctoral fellow in the laboratory of Dr. Howard Schulman at Stanford University, I continued investigations of voltage-gated channel regulation, shifting focus to calmodulin and Ca2+-calmodulin-dependent protein kinase II (CaMKII). Finally, I trained as an associate research scientist at Yale in the laboratory of Steve Waxman studying voltage-gated sodium channel regulation via calmodulin and Map kinases before starting my academic career.
Q: Could you provide some background on the Hudmon research group?A: For almost 30 years, our research has focused on kinase and ion channel regulation by calcium signaling; a topic of passion since my PhD training. Our independent research program began in 2006 in the Department of Biochemistry and Molecular Biology and Stark Neuroscience Research Institute at the Indiana University School of Medicine in Indianapolis. The laboratory moved in 2017 to Purdue University where we are located in the Department of Medicinal Chemistry and Molecular Pharmacology in the College of Pharmacy. We continue to study how calcium signaling effectors/modulators target ion channels and other proteins to regulate excitability in neurons and myocytes. Research in the Hudmon laboratory is motivated by trying to understand how key proteins like kinases and ion channels regulate the function of excitable cells in normal and pathological conditions like stroke, epilepsy and heart failure. Our laboratory employs molecular, biochemical and biophysical strategies to identify mechanisms regulating ion channels, kinases and their targets.
Q: How has the MultiPep improved your research?A: While I’ve utilized peptides throughout my research career, the early years primarily employed soluble purchased peptides in assays designed to identify protein levels or activity. We began using the SPOTs peptide approach to determine post-translational modifications and protein-protein interactions in the mid-2000s. Experimental successes using purchased immobilized peptide blots lead to obtaining an early generation MultiPep capable of synthesizing 1200 peptides on two cellulose membranes. This instrument is still in operation and splits peptide synthesis duty with a newer MultiPep capable of synthesizing 2400 peptides on 4 cellulose membranes. The ability to synthesize 100s of defined peptides has revolutionized our laboratory’s ability to quickly define phosphorylation and sites of interaction between proteins. The biological validity of the SPOTs approach ultimately requires cellular and in vivo studies, however, basic questions that previously required several months are completed in a fraction of the time using peptide arrays. We’ve published 9 articles using the MultiPep instruments in the SPOT format to identify mechanisms underlying protein regulation by protein-protein interactions and post-translational modifications. Array mapping of voltage-gated sodium channel (A) Frame-shift peptide array synthesis of protein (B) Mapping of peptides on protein domains (C) Identification of phosphorylation sites
For example, it can be quite tedious and difficult to identify phosphorylation sites on a large protein like a voltage-gated sodium channel (>200,000 Daltons) using traditional strategies like fusion protein analysis with mutagenesis and truncations. However, mapping the ion channel as continuous peptides to generate a SPOT array spanning the intracellular domains of the protein can be used to quickly identify mechanisms of interest. The identification of potential phosphorylation sites using in vitro kinase assays with recombinant kinase and ATP-γP32 can reveal potential phosphorylation sites that can be later tested with full protein mutagenesis to explore their biological relevance. The caveat that must be addressed is that array peptides from a protein may not be structurally relevant in the native folded protein and therefore one needs a functional assay (in our case whole-cell patch clamping) to identify the contribution of the post-translational modification to a protein’s function. We have also used the SPOTs approach to identify peptides that can be used to disrupt protein-protein interactions; peptides that become powerful reagents for both providing basic mechanisms underlying a protein’s regulation, as well as identifying potential therapeutic strategies whereby soluble peptides may be used to modulate a protein’s activity in cells and in vivo.