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G-protein-coupled receptors (GPCRs) are important therapeutic targets expressed on the cell surface. Here, we present a protocol for identifying physiologically relevant binding proteins of adhesion GPCR GPR110. We describe steps for in-cell chemical crosslinking, immunoprecipitation, and quantitative high-resolution mass spectrometry. Notably, we detail a label-free quantitation strategy that eliminates irrelevant interacting proteins using an inactive GPR110 mutant with impaired surface expression. Furthermore, we outline procedures for validating the identified partners.

Although PI3K/Akt signaling that regulates neuronal survival has been implicated in the deleterious effects of ethanol on the central nervous system, underlying molecular mechanisms have not been fully elucidated. Akt–membrane interaction is a prerequisite step for Akt activation since it induces interdomain conformational changes to an open conformer that allows Akt phosphorylation by upstream kinases. In this study, we investigated the effect of ethanol on Akt activation by quantitatively probing Akt conformation using chemical cross-linking, 18O labeling and mass spectrometry. We found that ethanol at pharmacologically relevant concentrations (20 or 170 mM) directly interacts with Akt and alters the local pleckstrin homology domain configuration near the PIP3-binding site. We also found that ethanol significantly impairs subsequent membrane-induced interdomain conformational changes needed for Akt activation. The observed alteration of Akt conformation caused by ethanol during the activation sequence provides a new molecular basis for the effects of ethanol on Akt signaling. The in vitroconformation-based approach employed in this study should also be useful in probing the molecular mechanisms for the action of ethanol or drugs on other signaling proteins, particularly for those undergoing dramatic conformational change during activation processes such as members of AGC kinase super family.

Akt, a serine/threonine kinase, plays a critical role in cell survival. Upon growth factor receptor stimulation, cytosolic Akt is recruited to the plasma membrane by phospholipid binding and activated through phosphorylation at Thr308and Ser473. Although crystal structures for the parts of Akt have been reported, neither the three-dimensional structure of the whole molecule nor sequential conformational changes during activation have been demonstrated. In this study, we demonstrated that Akt undergoes dramatic interdomain conformational changes during activation processes by probing the three-dimensional structure of full-length Akt in solution using chemical cross-linking and tandem mass spectrometry. The cross-linking results not only provided new structural information but also revealed distinctive spatial arrangements of individual domains in the Akt molecule in resting, membrane-interacted, phosphorylated, and substrate-bound states. Our data allowed a new model for stepwise interdomain conformational changes in Akt activation sequence, setting a stage for the further investigation on Akt-membrane, Akt-protein, and/or Akt-drug interactions in solution to understand molecular mechanisms involved in physiological and pathophysiological processes of cell survival.

Mass spectrometry with chemical cross-linking was used to probe the conformational changes of HSA (human serum albumin) in solution on interaction with monounsaturated OA (oleic acid) or polyunsaturated AA (arachidonic acid) or DHA (docosahexaenoic acid). Fatty acid-free or -bound HSA was modified with lysine-specific cross-linkers and digested with trypsin. Cross-linked peptides were analysed by nano-electrospray ionization MS to localize the sites of cross-linking. Our data indicated that a local conformational change involving movement of the side chains of Lys-402 of subdomain IIIA or Lys-541 of subdomain IIIB occurred upon binding of all three fatty acids. Our data also indicated that the side chains of Lys-205 (IIA) and Lys-466 (IIIA) moved closer towards each other upon binding AA or DHA, but not OA, suggesting that the conformations of HSA when bound to mono- and poly-unsaturated fatty acids are distinctively different. While these observations agreed with previous X-ray crystallographic studies, the distances between ε-amino groups of most cross-linked lysine pairs were shorter than the crystal structure predicted, possibly reflecting a discrepancy between the solution and crystal structures. This method can serve as a useful complement to X-ray crystallography, particularly in probing the structure of a protein in solution.

Surface-enhanced Raman (SER) spectroelectrochemical studies were carried out to identify the surface species formed on Co–Cr–Mo alloy under different conditions. The surface films identified for the alloy in air include chromium(III) oxide, molybdate, molybdenum(VI) oxide and possibly cobalt(II) oxide. The air-formed molybdenum species disappear quickly in 0.15 M NaCl. In 0.15 M NaCl, part of the CoO and Cr2O3 might react with water to form Co(OH)2 and Cr(OH)3 , respectively. It is difficult to reduce these lower oxidation state oxides and hydroxides of Cr and Co. At high potentials, Co(OH)2 species remain at the interfaces. From electrochemical data, it appears that the alloy passivates very well, especially in artificial saliva. At lower potential, the Cr species play major roles in the passivation of the alloy, and at higher potential the Co species take on these roles. The dissolution rates of Mo from the Co–Cr–Mo alloy in NaCl and NaOH solutions are too low to be measured, indicating that the passive films of Cr and/or Co protect further oxidation of underlying Mo in the alloy. Copyright © 1999 John Wiley & Sons, Ltd.