Native mass spectrometry (nMS) is now widely applied to investigate non-covalently assembled biomolecule complexes. nMS requires the use of near-neutral pH and volatile buffers to preserve the native state of proteins. However, buffer exchange into nMS-compatible solvent is usually performed manually, which results in a time-consuming and tedious process, thus appearing as a major drawback for nMS analysis. Conversely, online coupling of size exclusion chromatography (SEC) to nMS affords a fast-automated and improved desalting, but also provides an additional dimension of separation for complex protein mixtures. We illustrate here the benefits of SEC-nMS compared to manual offline desalting for the characterization of a wide variety of biological systems, ranging from multiprotein assemblies, protein-ligands and protein-nucleic acid complexes, to proteins in a detergent environment. We then highlight the potential of the coupling to further integrate ion mobility while preserving the native conformations of proteins, allowing for rapid collision cross section measurement and even collision-induced unfolding experiments. Finally, we show that online SEC coupling can also serve as the basis for multidimensional non-denaturing liquid chromatography (LC) workflows, with the SEC acting as a fast desalting device, helping to achieve first dimension LC separation in optimal chromatographic conditions while being compatible with further nMS analysis.

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EPAC1, a cAMP-activated GEF for Rap GTPases, is a major transducer of cAMP signaling and a therapeutic target in cardiac diseases. The recent discovery that cAMP is compartmentalized in membrane-proximal nanodomains challenged the current model of EPAC1 activation in the cytosol. Here, we discover that anionic membranes are a major component of EPAC1 activation. We find that anionic membranes activate EPAC1 independently of cAMP, increase its affinity for cAMP by two orders of magnitude, and synergize with cAMP to yield maximal GEF activity. In the cell cytosol, where cAMP concentration is low, EPAC1 must thus be primed by membranes to bind cAMP. Examination of the cell-active chemical CE3F4 in this framework further reveals that it targets only fully activated EPAC1. Together, our findings reformulate previous concepts of cAMP signaling through EPAC proteins, with important implications for drug discovery.

In an effort to provide an overview of the biophysical approaches used to study G-protein-coupled receptors, we chose to consider the adenosine A2A receptor as a model, as it is widely reported in the literature to explore the way GPCRs are studied nowadays. After a brief introduction of the receptor, we gathered descriptions of the various tools used to investigate the pharmacology and structure of the A2A receptor. We began by describing the key developments which have led to successful studies of GPCRs including the cloning, expression and purification of A2A, and the subsequent characterizations including quality control, binding and functional studies that have been necessary for the further understanding of the receptor. Then, we reviewed the reconstitution of A2A into nanodiscs as well as the use of this biological material in structural mass spectrometry, NMR, calorimetry and various other approaches to gain not only information about the structure and function of A2A, but also the dynamics of the receptor and the tools necessary to pursue such investigations. The body of techniques presented herein are applicable to all GPCRs amenable to purification.