Method for Identifying Nonspecific Protein−Protein Interactions in Nanoelectrospray Ionization Mass Spectrometry

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The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein-ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.

Section: Nonspecific Bindingmentioning

confidence: 99%

Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

Kitova

El-Hawiet

Schnier

et al. 2012

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“…Nanospray [34 -36] is often a gentle ionization method of allowing the use of less harsh conditions including a lower heated capillary temperature, the absence of heater gas, and a lower spray voltage while also providing high ionization efficiency for small-sample consumption. These advantages favor the application of nanospray to study protein conformation and noncovalent complexes [37][38][39].…”

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confidence: 99%

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

Guo

Zhang

Song

et al. 2009

Coldspray ionization (CSI) mass spectrometry, a variant of electrospray ionization (ESI) operating at low temperature (20 to Ϫ80°C), has been used to characterize protein conformation and noncovalent complexes. A comparison of CSI and ESI was presented for the investigation of the equilibrium acid-induced unfolding of cytochrome c, ubiquitin, myoglobin, and cyclophilin A (CypA) over a wide range of pH values in aqueous solutions. CSI and nanoelectrospray ionization (nanoESI) were also compared in their performance to characterize the conformational changes of cytochrome c and myoglobin. Significant differences were observed, with narrower charged-state distribution and a shift to lower charge state in the CSI mass spectra compared with those in ESI and nanoESI mass spectra. The results suggest that CSI is more prone to preserving folded protein conformations in solution than the ESI and nanoESI methods. Moreover, the CSI-MS data are comparable with those obtained by other established biophysical methods, which are generally acknowledged to be the suitable techniques for monitoring protein conformation in solution. Noncovalent complexes of holomyoglobin and the protein-ligand complex between CypA and cyclosporin A (CsA) were also investigated at a neutral pH using the CSI-MS method. The results of this study suggest the ability of CSI-MS in retaining of protein conformation and noncovalent interactions in solution and probing subtle protein conformational changes. Additionally, the CSI-MS method is capable of analyzing quantitatively equilibrium unfolding transitions of proteins. CSI-MS may become one of the promising techniques for investigating protein conformation and noncovalent protein-ligand interactions in solution. C haracterization of native conformations and noncovalent complexes of proteins is of great significance to understanding a variety of biological processes at the molecular level. Electrospray ionization mass spectrometry (ESI-MS) [1, 2] has grown into a powerful technique in this field. Besides its capability of handling large biomolecules, the advantages of ESI-MS over other spectroscopic and biophysical methods include high analysis speed, minimal sample consumption, and the characterization of individual conformational states that may coexist in solution at equilibrium [3][4][5]. The charge-state distribution (CSD) in the ESI mass spectra represents a specific conformational state of protein [6 -9]. Broad CSDs at high charge states are generally associated with unfolded proteins, whereas narrower distributions centered on lower charge states are treated as characteristics of folded proteins [6, 10 -15]. However, the harsh conditions during the ionization process in MS are often detrimental to the preservation of protein conformation and the survival of noncovalent complexes. Many studies have examined the influence of the ionization process and the operating settings of ESI, including the curtain gas [16,17], the pressure in the ion source [18 -20], the temperature [21][22][23][24][25][26],...

“…Destabilization of proteins may occur in the absence of water if water is preferentially involved in noncovalent interactions [3]. In some cases, protein-ligand complexes partially dissociate in the gas phase [4][5][6] and in other cases, nonspecific adducts or clusters, which are artefacts of mass spectrometry and do not represent solution species, can be formed [5,7,8]. Thus, the binding in gas-phase noncovalent complexes will not necessarily parallel solution binding.…”

Section: Introductionmentioning

confidence: 99%

“…[Absolute binding energies of gas-phase complexes can be measured with blackbody infrared radiative dissociation (BIRD) experiments (see for example [5,8,[11][12][13])].…”

Section: Introductionmentioning

confidence: 99%

Solution and Gas-Phase H/D Exchange of Protein–Small-Molecule Complexes: Cex and Its Inhibitors

Kang

Terrier

Ding

et al. 2011

The properties of noncovalent complexes of the enzyme exo-1,4-β-D-glycanase ("Cex") with three aza-sugar inhibitors, deoxynojirimycin (X 2 DNJ), isofagomine lactam (X 2 IL), and isofagomine (X 2 IF), have been studied with solution and gas-phase hydrogen deuterium exchange (H/Dx) and measurements of collision cross sections of gas-phase ions. In solution, complexes have lower H/Dx levels than free Cex because binding the inhibitors blocks some sites from H/Dx and reduces fluctuations of the protein. In mass spectra of complexes, abundant Cex ions are seen, which mostly are formed by dissociation of complexes in the ion sampling interface. Both complex ions and Cex ions formed from a solution containing complexes have lower cross sections than Cex ions from a solution of Cex alone. This suggests the Cex ions formed by dissociation "remember" their solution conformations. For a given charge, ions of the complexes have greater gas-phase H/Dx levels than ions of Cex. Unlike cross sections, H/Dx levels of the complexes do not correlate with the relative gas-phase binding strengths measured by MS/MS. Cex ions from solutions with or without inhibitors, which have different cross sections, show the same H/Dx level after 15 s, indicating the ions may fold or unfold on the seconds time scale of the H/Dx experiment. Thus, cross sections show that complexes have more compact conformations than free protein ions on the time scale of ca. 1 ms. The gas-phase H/Dx measurements show that at least some complexes retain different conformations from the Cex ions on a time scale of seconds.