cytochrome-c-t and ammonium-acetate

Electro-osmotically induced Joule heating in theta tips and its effect on protein denaturation were investigated. Myoglobin, equine cytochrome c, bovine cytochrome c, and carbonic anhydrase II solutions were subjected to electro-osmosis in a theta tip and all of the proteins were denatured during the process. The extent of protein denaturation was found to increase with the applied square wave voltage and electrolyte concentration. The solution temperature at the end of a theta tip was measured directly by Raman spectroscopy and shown to increase with the square wave voltage, thereby demonstrating the effect of Joule heating through an independent method. The electro-osmosis of a solution comprised of myoglobin, bovine cytochrome c, and ubiquitin demonstrated that the magnitude of Joule heating that causes protein denaturation is positively correlated with protein melting temperature. This allows for a quick determination of a protein's relative thermal stability. This work establishes a fast, novel method for protein conformation manipulation prior to MS analysis and provides a temperature-controllable platform for the study of processes that take place in solution with direct coupling to mass spectrometry. Graphical Abstract ᅟ.

Topics: Acetates; Animals; Cattle; Cytochromes c; Heating; Horses; Myoglobin; Protein Denaturation; Proteins; Spectrometry, Mass, Electrospray Ionization; Spectrum Analysis, Raman; Transition Temperature; Ubiquitin

Factors that influence the charging of protein ions formed by electrospray ionization from aqueous solutions in which proteins have native structures and function were investigated. Protein ions ranging in molecular weight from 12.3 to 79.7 kDa and pI values from 5.4 to 9.6 were formed from different solutions and reacted with volatile bases of gas-phase basicities higher than that of ammonia in the cell of a Fourier-transform ion cyclotron resonance mass spectrometer. The charge-state distribution of cytochrome c ions formed from aqueous ammonium or potassium acetate is the same. Moreover, ions formed from these two solutions do not undergo proton transfer to 2-fluoropyridine, which is 8 kcal/mol more basic than ammonia. These results provide compelling evidence that proton transfer between ammonia and protein ions does not limit protein ion charge in native electrospray ionization. Both circular dichroism and ion mobility measurements indicate that there are differences in conformations of proteins in pure water and aqueous ammonium acetate, and these differences can account for the difference in the extent of charging and proton-transfer reactivities of protein ions formed from these solutions. The extent of proton transfer of the protein ions with higher gas-phase basicity bases trends with how closely the protein ions are charged to the value predicted by the Rayleigh limit for spherical water droplets approximately the same size as the proteins. These results indicate that droplet charge limits protein ion charge in native mass spectrometry and are consistent with these ions being formed by the charged residue mechanism. Graphical Abstract ᅟ.

Topics: Acetates; Ammonia; Circular Dichroism; Cytochromes c; Ion Mobility Spectrometry; Molecular Weight; Proteins; Protons; Pyridines; Solutions; Spectrometry, Mass, Electrospray Ionization; Water

The effectiveness of two new supercharging reagents for producing highly charged ions by electrospray ionization (ESI) from aqueous solutions in which proteins have native structures and reactivities were investigated. In aqueous solution, 2-thiophenone and 4-hydroxymethyl-1,3-dioxolan-2-one (HD) at a concentration of 2% by volume can increase the average charge of cytochrome c and myoglobin by up to 163%, resulting in even higher charge states than those that are produced from water/methanol/acid solutions in which these proteins are denatured. The greatest extent of supercharging occurs in pure water, but these supercharging reagents are also highly effective in aqueous solutions containing 200 mM ammonium acetate buffer commonly used in native mass spectrometry (MS). These reagents are less effective supercharging reagents than m-nitrobenzyl alcohol (m-NBA) and propylene carbonate (PC) when ions are formed from water/methanol/acid. The extent to which loss of the heme group from myoglobin occurs is related to the extent of supercharging. Results from guanidine melts of cytochrome c monitored with tryptophan fluorescence show that the supercharging reagents PC, sulfolane and HD are effective chemical denaturants in solution. These results provide additional evidence for the role of protein structural changes in the electrospray droplet as the primary mechanism for supercharging with these reagents in native MS. These results also demonstrate that for at least some proteins, the formation of highly charged ions from native MS is no longer a significant barrier for obtaining structural information using conventional tandem MS methods.

Topics: Acetates; Benzyl Alcohols; Cytochromes c; Indicators and Reagents; Ions; Methanol; Myoglobin; Propane; Protein Conformation; Proteins; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry; Tandem Mass Spectrometry; Thermodynamics; Thiophenes; Water

Mass Spectra of charge states of folded proteins were obtained with nanospray and aqueous solution containing 20 microM the protein (ubiquitin, cytochrome c, lysozyme) and one of the NaA salts NaCl, NaI, NaAc (acetate) (1-10 mM). At very low collision activated decomposition (CAD), the mass spectra of a protein with charge z exhibited a replacement of zH+ with zNa+ and also multiple adducts of NaA. Higher CAD converts the NaA adduct peaks to Na minus H peaks. These must be due to loss of HA where the H was provided by the protein. The degree of HA loss with increasing CAD followed the order I < Cl < Ac. Significantly, the intensity of the ions with n (Na minus H) adducts showed a downward break past an n(MAX) which is equal to the number of acidic residues of the protein plus the charge of the protein. All the observations could be rationalized within the framework of the electrospray mechanism and the charge residue model, which predict that due to extensive evaporation of solvent, the solutes will reach very high concentrations in the final charged droplets. At such high concentrations, positive ions such as Na+, NH4+ form ion pairs with ionized acidic residues and the negative A- form ion pairs with ionized basic residues of the protein. Adducts of Na+, and NaA to backbone amide groups occur also. This reaction mechanism fits all the experimental observations and provides predictions that the number of acidic and basic groups at the surface of the gaseous protein that remain ionized can be controlled by the absence or presence of additives to the solution.

Topics: Acetates; Acetic Acid; Amino Acids, Acidic; Amino Acids, Basic; Cytochromes c; Ions; Muramidase; Particle Size; Proteomics; Sodium Acetate; Sodium Chloride; Sodium Iodide; Spectrometry, Mass, Electrospray Ionization; Ubiquitin

The decrease in the sensitivity of electrospray ionization mass spectrometry caused by the presence of metal salts, such as sodium chloride, in the sample matrix is well known and is particularly problematic for biological samples. We report here that addition of high levels of ammonium acetate can improve analyte signal in aqueous electrospray solutions and counteracts the signal suppression caused by sodium chloride. A approximately 3-fold improvement in S/N is obtained by adding 8 M ammonium acetate to aqueous solutions of cytochrome c without added sodium chloride. No organic solvents or acids are added into the electrospray solutions. The signal-to-noise ratios of cytochrome c and ubiquitin (10(-)(5) M) ions formed from aqueous solutions containing 2.0 x 10(-)(2) M sodium chloride are improved by factors of approximately 7 and 11, respectively, by adding 7 M ammonium acetate to the solution. We propose that this effect is a result of the precipitation of Na(+) and Cl(-) from solution within the evaporating electrospray droplets prior to the formation of gas-phase protein ions. This method is potentially useful for improving the abundance of protein ions formed from solutions in which the molecules have a nativelike conformation and is particularly advantageous for such solutions that have high levels of sodium.

Topics: Acetates; Buffers; Cytochromes c; Signal Processing, Computer-Assisted; Sodium Chloride; Spectrometry, Mass, Electrospray Ionization; Ubiquitin