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

Abstract

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],...

Air flow assisted ionization for remote sampling of ambient mass spectrometry and its application

“…The conformational transition of cytochrome c is very sensitive to changes in pH [34] and, along with further acidification of the solution, the distribution and maximum charge states are gradually shifted to higher charge states. [35] The phenomena observed here may be explained by desolvation being assisted by the high extracting air flow rate inducing a minor decrease in pH of the charged droplets, resulting in a shift to higher charge states for cytochrome c. The results obtained here further demonstrated that ion formation in AFAI is promoted by high air flow rates due to enhanced desolvation of charged droplets.…”

“…The charge‐state distributions of cytochrome c using AFA‐ESI were lower than those obtained using normal ESI. The conformational transition of cytochrome c is very sensitive to changes in pH34 and, along with further acidification of the solution, the distribution and maximum charge states are gradually shifted to higher charge states 35. The phenomena observed here may be explained by desolvation being assisted by the high extracting air flow rate inducing a minor decrease in pH of the charged droplets, resulting in a shift to higher charge states for cytochrome c .…”

High pressure nanoelectrospray ionization mass spectrometry for analysis of aqueous solutions

“…The results here are consistent with other previous reports that use pneumatic-assisted electrospray, nanoESI and cold-ESI for native proteins. 10,[24][25][26][27][28][29][30][31] The reduced charge state is due to the reduced number of acidic or basic sites available on the outer surface of the tightly folded protein structure. Ionization under high pressure was also found to be gentle.…”

Advances in MS Based Strategies for Probing Ligand-Target Interactions: Focus on Soft Ionization Mass Spectrometric Techniques

“…Besides, the removal of the solvent in the solution may cause the difference of the conformation of certain non-covalent protein-ligand complexes between the liquid state and gas phase of the mass spectrometer, and thus it is not suitable for some highly complicated protein-ligand complexes with thermal properties. In order to better maintain the natural conformation of proteins in solution, researchers have made some improvements to the ESI source, including electrosonic spray ionization (Wiseman et al, 2004) and cold spray mass spectrometry (CSI-MS) (Guo et al, 2009). By increasing the polarizabilities of the molecules by cooling the liquid spray and the solvent removal process, CSI-MS could promote the ionization of molecules and has been successfully applied to the study of supra-molecular systems that are heat unstable and cannot be analyzed by ESI-MS.…”