Abstract
Proteins and small molecules are the effectors of physiological action in biological systems and comprehensive methods are needed to analyze their modifications, expression levels and interactions. Systems-scale characterization of the proteome requires thousands of components in high-complexity samples to be isolated and simultaneously probed. While protein microarrays offer a promising approach to probe systems-scale changes in a high-throughput format, they are limited by the need to individually synthesize tens of thousands of proteins. We present an alternative technique, which we call diffusive gel (DiG) stamping, for patterning a microarray using a cellular lysate enabling rapid visualization of dynamic changes in the proteome as well protein interactions. A major advantage of the method described is that it requires no specialized equipment or in-vitro protein synthesis, making it widely accessible to researchers. The method can be integrated with mass spectrometry, allowing for the discovery of novel protein interactions. Here, we describe and characterize the sensitivity and physical features of DiG-Stamping. We demonstrate the biologic utility of DiG-Stamping by (1) identifying the binding partners of a target protein within a cellular lysate and by (2) visualizing the dynamics of proteins with multiple post-translational modifications.
Highlights
Systems-scale analysis of the proteome, for example the identification of binding partners or post-translational modifications, requires thousands of components in high-complexity samples to be isolated and simultaneously probed
We have developed a novel method, diffusive gel (DiG)-Stamping, that allows for biologically-motivated complexity reduction in high-content samples and subsequent unbiased discovery of novel protein interactions and proteome dynamics
Methods for the analysis of novel proteins and small molecule interactions are essential for the study of biological systems
Summary
Systems-scale analysis of the proteome, for example the identification of binding partners or post-translational modifications, requires thousands of components in high-complexity samples to be isolated and simultaneously probed. The equipment needed for fabricating microarrays can restrict their use, novel approaches such as patterning with an agarose-based stamp have been implemented [6]. Notwithstanding these advantages, the manufacture and use of protein microarrays has been significantly hindered by the need to synthesize thousands of partial or fulllength proteins. The method presented here addresses these limitations by patterning an array using a cellular lysate without the need for any specialized lab equipment, enabling rapid visualization of the proteome and protein interactions
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