Biomedical subjects

Gavin MacBeath

Publications and source records attributed to Gavin MacBeath.

7 recordsLinked to original sources

PubMed2006

Circumventing the problems caused by protein diversity in microarrays: implications for protein interaction networks.

Protein microarrays provide a well-controlled, high-throughput way to uncover protein-protein interactions. One problem with this and other standardized assays, however, is that proteins vary considerably with respect to their physical properties. If a simple threshold-based approach is used to define protein-protein interactions, the resulting binary networks can be strongly biased. Here, we investigate the extent to which even closely related protein interaction domains vary when printed as microarrays. We find that, when a collection of well behaved, monomeric Src homology 2 (SH2) domains are printed at the same concentration, they vary by up to 50-fold with respect to the resulting surface density of active protein. When a threshold-based binding assay is performed on these domains using fluorescently labeled phosphopeptides, a misleading picture of the underlying biophysical interactions emerges. This problem can be circumvented, however, by obtaining saturation binding curves for each protein-peptide interaction. Importantly, the equilibrium dissociation constants obtained from these curves are independent of the surface density of active protein. We submit that an increased emphasis should be placed on obtaining quantitative information from protein microarrays and that this should serve as a more general goal in all efforts to define large-scale protein interaction networks.

Biosensing Techniques↗

PubMed2006

Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays.

One of the principal challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach--compatible with genome-wide investigations--that enables us to measure the equilibrium dissociation constant (K(D)) of recombinant PDZ domains for fluorescently labeled peptides that represent physiologically relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the K(D) for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (K(D) < or = 10 microM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network.

Amino Acid Sequence↗

PubMed2006

State-based discovery: a multidimensional screen for small-molecule modulators of EGF signaling.

As an alternative to conventional, target-oriented drug discovery, we report a strategy that identifies compounds on the basis of the state that they induce in a signaling network. Immortalized human cells are grown in microtiter plates and treated with compounds from a small-molecule library. The target network is then activated and lysates derived from each sample are arrayed onto glass-supported nitrocellulose pads. By probing these microarrays with antibodies that report on the abundance or phosphorylation state of selected proteins, a global picture of the target network is obtained. As proof of concept, we screened 84 kinase and phosphatase inhibitors for their ability to induce different states in the ErbB signaling network. We observed functional connections between proteins that match our understanding of ErbB signaling, indicating that state-based screens can be used to define the topology of signaling networks. Additionally, compounds sort according to the multidimensional phenotypes they induce, suggesting that state-based screens may inform efforts to identify the targets of biologically active small molecules.

Antibodies↗

PubMed2006

Collecting and organizing systematic sets of protein data.

Systems biology, particularly of mammalian cells, is data starved. However, technologies are now in place to obtain rich data, in a form suitable for model construction and validation, that describes the activities, states and locations of cell-signalling molecules. The key is to use several measurement technologies simultaneously and, recognizing each of their limits, to assemble a self-consistent compendium of systematic data.

Animals↗

PubMed2005

A quantitative protein interaction network for the ErbB receptors using protein microarrays.

Although epidermal growth factor receptor (EGFR; also called ErbB1) and its relatives initiate one of the most well-studied signalling networks, there is not yet a genome-wide view of even the earliest step in this pathway: recruitment of proteins to the activated receptors. Here we use protein microarrays comprising virtually every Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain encoded in the human genome to measure the equilibrium dissociation constant of each domain for 61 peptides representing physiological sites of tyrosine phosphorylation on the four ErbB receptors. This involved 77,592 independent biochemical measurements and provided a quantitative protein interaction network that reveals many new interactions, including ones that fall outside of our current view of domain selectivity. By slicing through the network at different affinity thresholds, we found surprising differences between the receptors. Most notably, EGFR and ErbB2 become markedly more promiscuous as the threshold is lowered, whereas ErbB3 does not. Because EGFR and ErbB2 are overexpressed in many human cancers, our results suggest that the extent to which promiscuity changes with protein concentration may contribute to the oncogenic potential of receptor tyrosine kinases, and perhaps other signalling proteins as well.

Cell Line↗

PubMed2003

Profiling receptor tyrosine kinase activation by using Ab microarrays.

Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Understanding these networks at a circuit level requires devices to measure the amounts and activities of multiple proteins in a rapid and accurate manner. Ab microarrays have previously been applied to the quantification of labeled recombinant proteins and proteins in serum. The development of methods to analyze intracellular signaling molecules on microarrays would make Ab arrays widely useful in systems biology. Here we describe the fabrication of multiplex Ab arrays sensitive to the amounts and modification states of signal transduction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microarrays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratiometric microarrays correlate well with data obtained by using traditional approaches, but the arrays are faster and simpler to use. The integration of microplate and microarray methods for crude cell lysates should make it possible to identify and analyze small molecule inhibitors of signal transduction processes with unprecedented speed and precision. We demonstrate the future potential of this approach by characterizing the action of the epidermal growth factor receptor inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based screening in 96- and 384-well plates should allow small molecules to be identified with specific inhibitory profiles against a signaling network.

Animals↗

PubMed2002

Protein microarrays and proteomics.

The system-wide study of proteins presents an exciting challenge in this information-rich age of whole-genome biology. Although traditional investigations have yielded abundant information about individual proteins, they have been less successful at providing us with an integrated understanding of biological systems. The promise of proteomics is that, by studying many components simultaneously, we will learn how proteins interact with each other, as well as with non-proteinaceous molecules, to control complex processes in cells, tissues and even whole organisms. Here, I discuss the role of microarray technology in this burgeoning area.

Forecasting↗