title: Interpreting conformational effects in protein nano-ESI-MS spectra.
abstract: 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.
authors.affiliation: Institute of Chemistry, Johannes Kepler University, Altenbergerstrasse 69, 4040 Linz, Austria.
authors.forename: Maria
authors.initials: M
authors.lastname: Samalikova
authors.forename: Irena
authors.initials: I
authors.lastname: Matecko
authors.forename: Norbert
authors.initials: N
authors.lastname: Müller
authors.forename: Rita
authors.initials: R
authors.lastname: Grandori
mesh_terms: D006863:Hydrogen-Ion Concentration; D036103:Nanotechnology / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D011506:Proteins; D012997:Solvents