Probing protein stabilization by glycerol using electrospray mass spectrometry

Grandori, Rita; Matecko, Irena; Mayr, Petra; Müller, Norbert

doi:10.1002/jms.193

Cited by 32 publications

(31 citation statements)

References 19 publications

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“…The envelope of the unfolded protein at pH 2.2 is centered on the peak of 17+ ion. These results are in agreement with previous measurements by regular3 or turbo‐ionspray7 ESI‐MS. Previous studies on the pH dependence of cyt c and lysozyme ESI mass spectra, in the presence of varying concentrations of glycerol as stabilizing agent, indicated that the main effect of pH changes in the range 4–2 is mediated by conformational changes 7.…”

Section: Resultssupporting

confidence: 93%

“…Cyt c undergoes a highly cooperative, acid‐induced unfolding transition between pH 3 and pH 2 5. The unfolded state at pH 2 has been characterized by several physico‐chemical methods, including circular dichroism (CD), fluorescence, small‐angle x‐ray scattering5, 6 and ESI‐MS 3, 7. These studies showed that the polypeptidic chain under these conditions loses most of its native tertiary and secondary structure.…”

Section: Introductionmentioning

confidence: 99%

“…Compact conformations typically result in protein envelopes shifted towards higher m / z values, as compared with unfolded conformations of the same molecule 32–36. We recently showed that ESI‐MS, in addition to discriminating folded from unfolded protein conformations, can monitor minor conformational changes, such as compression of protein structures induced by polyols and transition from the molten‐globule to the native state 7. ESI‐MS has also been applied to the study of the effect of different TFE concentrations on the oligomerization state of an amphipathic helix peptide 37.…”

Section: Introductionmentioning

confidence: 99%

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Uncoupled analysis of secondary and tertiary protein structure by circular dichroism and electrospray ionization mass spectrometry

2001

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Electrospray ionization mass spectrometry (ESI-MS) applied to protein conformational studies is a powerful new method that seems to provide specific information about protein tertiary structure. In this study, we analyzed the effect of trifluoroethanol (TFE) on a myoglobin peptide and cytochrome c (cyt c) at low pH by circular dichroism (CD) and ESI-MS. These experiments show that coil-to-helix transition per se does not affect ESI mass spectra, confirming that this technique is insensitive to the local conformation of the polypeptidic chain and, rather, reports on the tertiary contacts characterizing different protein conformations. This property makes ESI-MS an excellent method, complementary to CD, for the characterization of protein conformational changes. Fluorinated alcohols have been suggested to induce molten globule formation in acid-unfolded cyt c. The experiments described here show that TFE does not induce major changes in the ESI mass spectrum of cyt c at pH 2.2, indicating that no stabilization of compact, globular structures is detectable under the conditions employed. On the other hand, even low concentrations of TFE (2-5%) are shown to destabilize the folded state of the protein around the mid-point of its acid-induced unfolding transition.

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Section: Resultssupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Uncoupled analysis of secondary and tertiary protein structure by circular dichroism and electrospray ionization mass spectrometry

2001

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“…The strong pH dependence of protein stability is an obstacle to investigating the role of the pH on the ionization mechanism itself, uncoupled from conformational effects. The use of stabilizing agents, for example glycerol, has helped distinguish these two possible contributions to spectral changes in pH-variable experiments [26]. The results confirmed the predominance of conformational effects.…”

Section: Maria šAmalikova · Irena Matečko · Norbert Müller · Rita Grasupporting

confidence: 65%

Interpreting conformational effects in protein nano-ESI-MS spectra

et al. 2003

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Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is employed here to describe equilibrium protein conformational transitions and to analyze the influence of instrumental settings, pH, and solvent surface tension on the charge-state distributions (CSD). A first set of experiments shows that high flow rates of N(2) as curtain gas can induce unfolding of cytochrome c (cyt c) and myoglobin (Mb), under conditions in which the stability of the native protein structure has already been reduced by acidification. However, it is possible to identify conditions under which the instrumental settings are not limiting factors for the conformational stability of the protein inside ESI droplets. Under such conditions, equilibrium unfolding transitions described by ESI-MS are comparable with those obtained by other established biophysical methods. Experiments with the very stable proteins ubiquitin (Ubq) and lysozyme (Lyz) enable testing of the influence of extreme pH changes on the ESI process, uncoupled from acid-induced unfolding. When HCl is used for acidification, Ubq and Lyz mass spectra do not change between pH~7 and pH 2.2, indicating that the CSD is highly characteristic of a given protein conformation and not directly affected by even large pH changes. Use of formic or acetic acid for acidification of Ubq solutions results in major spectral changes that can be interpreted in terms of protein unfolding as a result of the increased hydrophobicity of the solvent. On the other hand, Lyz, cyt c, and Mb enable direct comparison of protein CSD (corresponding to either the folded or the unfolded protein) in HCl or acetic acid solutions at low pH. The values of surface tension for these solutions differ significantly. Confirming indications already present in the literature, we observe very similar CSD under these solvent conditions for several proteins in either compact or disordered conformations. The same is true for comparison between water and water-acetic acid for folded cyt c and Lyz. Thus, protein CSD from water-acetic solutions do not seem to be limited by the low surface tension of acetic acid as previously suggested. This result could reflect a general lack of dependence of protein CSD on the surface tension of the solvent. However, it is also possible that the effect of acetic acid on the precursor ESI droplets is smaller than generally assumed.

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“…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], the cone voltage [3,27], the spray mode [28], and the solvent composition [29,30]. To preserve the native protein conformation, a significant amount of effort has also been made on modifications to the ESI source.…”

mentioning

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

J. Am. Soc. Mass Spectrom.

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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],...

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Probing protein stabilization by glycerol using electrospray mass spectrometry

Cited by 32 publications

References 19 publications

Uncoupled analysis of secondary and tertiary protein structure by circular dichroism and electrospray ionization mass spectrometry

Uncoupled analysis of secondary and tertiary protein structure by circular dichroism and electrospray ionization mass spectrometry

Interpreting conformational effects in protein nano-ESI-MS spectra

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

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