Electrospray ionization of protein mixtures at low pH

Solutions composed of single proteins and mixtures of proteins are subjected to electrospray ionization to study the influence of protein components on the responses of one another. Protein matrix effects in electrospray ionization are particularly relevant to the development of top-down protein identification methodologies involving protein mixtures, whereby whole protein ions are subjected to tandem mass spectrometry. Emphasis is placed largely on solutions composed of equal parts methanol and water and 1% acetic acid. The results, therefore, are relevant to low-pH solutions with significant organic content, a commonly used set of conditions in electrospray ionization mass spectrometry that tends to denature proteins. Under these conditions, very similar response curves are measured for a variety of proteins after charge normalization. That is, when the data are plotted in terms of the concentration of charge sites, rather than in terms of the concentration of protein molecules, the slopes of the response curves as well as the point at which response becomes less than linear with concentration are similar. Charge normalization is made on the basis of the weighted average charge of a protein, as reflected in the electrospray ionization mass spectrum. When proteins can be regarded as a collection of equivalent charge sites, the signal response from one protein can be used to predict the responses for other proteins. Furthermore, it is also possible to predict the dependence of the signal response for a particular protein in a mixture on the concentration of other proteins in the mixture. Examining signal response on a weighted average charge basis appears to be an effective means for identifying situations in which the protein does not behave as a collection of equivalent charge sites.     

Analysis of high-density lipoprotein apolipoproteins recovered from specific immobilized pH gradient gel pI domains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The proteins associated with the circulating lipoproteins in the blood function not only for mediating lipid metabolism but also for maintaining structural stability of the micellelike structure. Any modifications of these proteins, by mutation or posttranslational modification, could compromise the function of these proteins and contribute to the development of cardiovascular disease. Because of the presence of extensive lipophilic domains, these proteins, when recovered from the lipoprotein particle (apolipoproteins) present an analytical challenge because of low solubility and proclivity toward aggregate formation. Our goal is to characterize these proteins by a combination of high-accuracy pI measurement coupled with MALDI analysis. In this report, we demonstrate the high resolution of immobilized pH gradient isoelectric focusing (IPG-IEF) for the analysis of these apolipoproteins isolated from serum HDL collected from a density gradient ultracentrifugation-based separation. The IPG separation of the HDL apolipoproteins was imaged and combined with digital analysis to produce a detailed pI profile of the apolipoproteins in the high-density lipoprotein (HDL) fraction. This is the first time that a high-resolution pI profile has been obtained for the HDL apolipoproteins. The feasibility of linking the pI profile to a MALDI-based molecular weight determination was achieved by incorporating passive elution of the intact proteins from the IPG gel with a four-component solvent mixture that solved the problem of recovery of the apolipoproteins from the IPG matrix. Twenty-five bands were observed in the pI profile. A survey analysis of 12 of these bands by MALDI indicated that they were associated with the known HDL apolipoproteins. While there is considerable overlap in the pI profiles of the apolipoproteins, linking the analysis with a MALDI-based second dimension in m/z is shown to be an efficient method to characterize this complex mixture of apolipoproteins.     

Estimates of protein surface areas in solution by electrospray ionization mass spectrometry

The extent of multiple charging of protein ions in electrospray ionization (ESI) mass spectra depends on the solvent-exposed surface area, but it may also be influenced by a variety of other extrinsic and intrinsic factors. Gas-phase ion chemistry (charge-transfer and charge-partitioning reactions) appears to be the major extrinsic factor influencing the extent of protonation as detected by ESI MS. In this work, we demonstrate that under carefully controlled conditions, which limit the occurrence of the charge-transfer reactions in the gas phase, charge-state distributions of protein ions can be used to assess the solvent-exposed surface area in solution. A set of proteins ranging from 5-kDa insulin to 500-kDa ferritin shows a clear correlation between the average charge in ESI mass spectra acquired under native conditions and their surface areas calculated based on the available crystal structures. An increase of the extent of charge-transfer reactions in the ESI interface results in a noticeable decrease of the average charge of protein ions across the entire range of tested proteins, while the charge-surface correlation is maintained. On the other hand, the intrinsic factors (e.g., a limited number of basic residues) do not appear to play a significant role in determining the protein ion charge. Based on these results, it is now possible to obtain estimates of the surface areas of proteins and protein complexes, for which crystal structures are not available. We also demonstrate how the ESI MS measurements can be used to characterize protein-protein interaction in solution by providing quantitative information on the subunit interfaces formed in protein associations.     

Effects of mobile-phase additives, solution pH, ionization constant, and analyte concentration on the sensitivities and electrospray ionization mass spectra of nucleoside antiviral agents

The effects of various mobile-phase additives, solution pH, pKa, and analyte concentration on electrospray ionization mass spectra of a series of purine and pyrimidine nucleoside antiviral agents were studied in both positive and negative ion models. The use of 1% acetic acid resulted in good HPLC separation and the greatest sensitivity for [M + H]+ ions. In the negative ion mode, 50 mM ammonium hydroxide gave the greatest sensitivity for [M - H]- ions. The sensitivities as [M + H]+ ions were significantly larger than the sensitivities as [M - H]- ions for purine antiviral agents. Vidarabine monophosphate and pyrimidine antiviral agents, however, showed comparable or greater sensitivities as [M - H]- ions. The sensitivity as [M + H]+ showed no systematic variation with pH; however, the sensitivity as [M - H]- did increase with increasing pH. At constant pH, the ion intensity of the protonated species increased with increasing pKa. At higher analyte concentrations, dimer (M2H+) and trimer (M3H+) ions were observed. [M + Na]+ adducts were the dominant ions with 0.5 mM sodium salts for these compounds. The spectra of the more basic purine antiviral agents showed no [M + NH4]+ adduct ions, but [M + NH4]+ ions were the major peaks in the spectra of the less basic pyrimidine antiviral agents with ammonium salts. The ammonium adduct ion was formed preferentially when the proton affinity of the analyte was close to that of NH3. Abundant [M + OAc]- ions were observed for all of the antiviral agents except vidarabine monophosphate from solutions with added HOAc, NaOAc, and NH4OAc. The utility of mobile phases containing 1% HOAc or 50 mM NH4OH was demonstrated for chromatographic separations.     

Accurate mass measurements for peptide and protein mixtures by using matrix-assisted laser desorption/ionization Fourier transform mass spectrometry

A new analytical scheme based on a combination of scanning FTMS, multiple-ion filling, and potential ramping methods has been developed for accurate molecular mass measurement of peptide and protein mixtures using broadband MALDI-FTMS. The scanning FTMS method alleviates the problems of time-of-flight effect for FTMS with an external MALDI ion source and provides a systematic means of sampling ions of different mass-to-charge ratios. The multiple-ion filling method is an effective way of trapping and retaining ions from successive ion generation/accumulation events. The potential ramping method allows the use of high trapping potentials for effective trapping of ions of high kinetic energies and the use of low trapping potentials for high-resolution detection of the trapped ions. With this analytical scheme, high-resolution broadband MALDI mass spectra covering a wide mass range of 1000-5700 Da were obtained. For peptide mixtures of mass range 1000-3500 Da, calibration errors of low part-per-millions were demonstrated using a parabolic calibration equation f2 = ML1/m2 + ML2/m + ML3, where f is the measured cyclotron frequency and ML1, ML2, and ML3 are calibration constants.     

Photodegradation study of sodium usnate solution: influence of pH

Photodegradation of 2.6 × 10^-5 M aqueous solutions of sodium usnate at various pH was studied. Photodegradation appeared to follow first-order kinetics and was found to be pH dependent. The degradation rate constant was calculated to be 9.20 × 10^-4 min^-1, 5.93 × 10^-4 min^-1, 9.69 × 10^-4 min^-1, and 9.88 × 10^-4 min^-1 at pH 6, pH 7, pH 8, and pH 9, respectively.     

A chemometric approach to detection and characterization of multiple protein conformers in solution using electrospray ionization mass spectrometry

Protein ion charge state distributions in electrospray ionization mass spectra have a potential to provide a wealth of information on protein dynamics, because they contain contributions from all protein conformers present in solution. Such ionic contributions often overlap, limiting the amount of useful information that can be extracted from the spectra. This difficulty is overcome in the present work by using a chemometric approach, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments are carried out by acquiring a series of spectra over a range of near-native and denaturing conditions to ensure significant changes in the conformers' populations. A total number of protein conformers sampled in the course of the experiments is determined by subjecting the set of collected spectra to singular value decomposition. Ionic contributions of each conformer to the total signal are then determined using a supervised optimization routine. Validation of the method has been carried out by monitoring acid- and alcohol-induced equilibrium states of well-characterized model proteins, chymotrypsin inhibitor 2 (two states), ubiquitin (three states) and apo-myoglobin (four states). For each of the model proteins, a new chemometric procedure yielded a picture of protein dynamics that was in excellent agreement with their documented behavior (as studied with other biophysical tools). The new method appears to be well-suited for monitoring protein dynamics in highly heterogeneous systems consisting of multiple proteins sampling a range of conformational states.     

Extended-range spectroscopic pH measurement using optimized mixtures of dyes

The spectroscopic technique for pH measurement is a well-established laboratory technique that can give high-accuracy pH values. Recent studies have shown the advantage of this technique over standard potentiometric methods for pH measurements in fresh water and seawater and also at high temperatures and pressures. However, a limitation of the spectroscopic technique is that a single pH dye is sensitive only over a narrow pH range. We have developed optimized dye mixtures that are both sensitive and accurate over a broad pH range. The measurement is robust and simple, requires a minimum of two wavelengths, and is independent of the volume of the dye mixture added. Optimization of the dye mixture formulation to maximize accuracy in a broad range of pH involves varying both the dye type and its mole fraction and also accounting for spectral noise. This technique has been successfully applied for in situ pH measurements of oilfield formation waters.     

Analyte incorporation and ionization in matrix-assisted laser desorption/ionization visualized by pH indicator molecular probes.

Despite the spreading applications of matrix-assisted laser desorption/ionization (MALDI), its fundamental understanding is still limited and under constant debate. This report focuses on the initial state of the analyte in the host matrix. pH indicator dyes serve as molecular probes since their color directly indicates their (de)protonation state. For a set of matrixes at their intrinsic pH, solution color was maintained, delivering clear proof for analyte incorporation in the solution charge state. Moreover, substantial solvent inclusion is determined by 1H NMR spectroscopy. MALDI mass spectra show a clear correlation to the dye charge state. However, the dominant solution species are not observed exclusively in the mass spectra, pointing to a proton transfer or proton neutralization activity of the matrix.     

Comprehensive on-line characterization of complex gas mixtures by quasi-simultaneous resonance-enhanced multiphoton ionization, vacuum-UV single-photon ionization, and electron impact ionization in a time-of-flight mass spectrometer: setup and instrument characterization

This paper reports on a newly developed mobile mass spectrometer for comprehensive on-line analysis of complex gas mixtures such as ambient air or industrial process gases. Three ionization methods, namely, the resonance-enhanced multiphoton ionization (REMPI), vacuum-ultraviolet single-photon ionization (SPI), and electron impact ionization (EI) are implemented in this instrument and can be operated (quasi-) simultaneously. By means of this setup, a wide range of compounds can be analyzed due to the unique ionization selectivitiy and sensitivity profiles provided by the different ionization techniques. The mass spectrometer is designed for field application even under severe conditions. The REMPI technique is suitable for the selective and soft ionization (without fragmentation) of aromatic compounds at trace level (ppbv/pptv). The also soft but less selective SPI technique with 118-nm vacuum-ultraviolet laser pulses is used as a second laser-based ionization method. Mass spectra obtained by this technique show profiles of most organic compounds (aliphatic and aromatic species) and of some low IP inorganic substances (e.g., ammonia, nitrogen oxide) down to ppbv concentrations. In addition to the laser-based ionization techniques, EI ionization can be used for analysis of the bulk components such as water, oxygen, nitrogen, and carbon dioxide as well as for detection of inorganic minor components such as HCN or HCl from combustion flue gases at ppmv concentration levels. Each method yields specific mass spectrometric information of the sample composition. Special techniques have been developed to combine the three ionization methods in a single mass spectrometer and to allow the quasi-parallel application of all three ionization techniques.     

Foaming binary solution mixtures of low molecular surfactant and polyelectrolyte

The lifetime of water solution foams of sodium dodecylsulfate (DDS, low molecular weight surfactant) and sodium carboxymethylcellulose (SCMC, polyelectrolyte) and their binary mixtures was experimentally investigated. The effects of ionic strength and acidity on the foam life were also determined. In binary solutions, a synergic effect of DDS and SCMC on the surface tension reduction, most likely resulting from the interaction of the surfactant with polymer, was found. The addition of NaCl into solution or increasing the ionic strength was found to decrease the surface tension and reduce interfacial mobility, hence increased foam lifetime. The relatively low lifetime of binary solution foams in acidic medium was attributed to the reaction between SCMC and acid, which resulted in relatively small reductions in the viscosity and consequently lowered the solution viscosity.     

A mobile mass spectrometer for comprehensive on-line analysis of trace and bulk components of complex gas mixtures: parallel application of the laser-based ionization methods VUV single-photon ionization, resonant multiphoton ionization, and laser-induced electron impact ionization

A newly developed compact and mobile time-of-flight mass spectrometer (TOFMS) for on-line analysis and monitoring of complex gas mixtures is presented. The instrument is designed for a (quasi-)simultaneous application of three ionization techniques that exhibit different ionization selectivities. The highly selective resonance-enhanced multiphoton ionization (REMPI) technique, using 266-nm UV laser pulses, is applied for selective and fragmentationless ionization of aromatic compounds at trace levels (parts-per-billion volume range). Mass spectra obtained using this technique show the chemical signature solely of monocyclic (benzene, phenols, etc.) and polycyclic (naphthalene, phenathrene, indol, etc.) aromatic species. Furthermore, the less selective but still fragmentationless single photon ionization (SPI) technique with 118-nm VUV laser pulses allows the ionization of compounds with an ionization potential below 10.5 eV. Mass spectra obtained using this technique show the profile of most organic compounds (aliphatic and aromatic species, like nonane, acetaldehyde, or pyrrol) and some inorganic compounds (e.g., ammonia, nitrogen monoxide). Finally, the nonselective ionization technique laser-induced electron-impact ionization (LEI) is applied. However, the sensitivity of the LEI technique is adjusted to be fairly low. Thus, the LEI signal in the mass spectra gives information on the inorganic bulk constituents of the sample (i.e., compounds such as water, oxygen, nitrogen, and carbon dioxide). Because the three ionization methods (REMPI, SPI, LEI) exhibit largely different ionization selectivities, the isolated application of each method alone solely provides specific mass spectrometric information about the sample composition. Special techniques have been developed and applied which allow the quasi-parallel use of all three ionization techniques for on-line monitoring purposes. Thus, a comprehensive characterization of complex samples is feasible jointly using the characteristic advantages of the three ionization techniques. Laboratory applications show results on rapid overview characterization of mineral oil-based fuels and coffee headspace. The first reported field applications include timely resolved on-line monitoring results on automobile exhausts and of waste incineration flue gas.     

Manipulation of protein charge states through continuous flow-extractive desorption electrospray ionization: a new ambient ionization technique

Manipulation of protein charge states in electrospray ionization-mass spectrometry (ESI-MS) has implications for the study of intact proteins, protein-protein interactions, post-translational modifications, and protein sequencing. Control of these protein charge states is often difficult to achieve with conventional methods of analysis. A novel ambient ionization configuration, continuous flow-extractive desorption electrospray ionization (CF-EDESI), is presented as a means to control the charge state distribution of proteins. A key feature of the CF-EDESI technique is the continuous flow needle, which is a hypodermic needle presented orthogonal to the electrospray source and delivers a solvent flow containing analytes for extractive desorption ionization. With this source design, the successful manipulation of cytochrome c and lysozyme charge states with the use of different additives, such as acetic acid and sulfolane, was demonstrated. Results were compared to data obtained with conventional electrospray ionization. Good agreement with previously reported studies of cytochrome c unfolding/folding studies, performed by conventional ESI-MS, is evident. In addition to the protein analysis presented, the CF-EDESI-MS technique should be applicable for analyzing atypical analyte and solvent systems by mass spectrometry while maintaining optimal electrospray source conditions.     

Parallel ion parking of protein mixtures

The multiple charging phenomenon resulting from electrospray ionization of proteins, while useful for the ability to make several mass measurements on a single component, can lead to highly complex spectra when mixtures are analyzed, as each component can generate multiple ions of distinct mass-to-charge ratio. Ion/ion proton-transfer reactions can overcome this problem by reduction of all components to the +1 charge state, but this typically requires the ability to extend the mass range of the instrument well beyond that available in most commercial instruments. Furthermore, reduction of protein charge to +1 also results in a reduction in detector response. Here it is shown that application of a relatively high amplitude, low-frequency auxiliary ac signal to the end cap electrodes of a 3-D ion trap during an ion/ion reaction can slow the ion/ion reaction rates of ions over a broad m/z range, in a process termed HALF parallel ion parking. Adjustment of the frequency and amplitude of the applied voltage allows the mass range into which the initial ion signal is moved to be controlled, allowing for the simplification of multicomponent mixtures within a mass range that is more commonly available on commercial systems. In addition to decreasing spectral complexity, this is advantageous for mixtures with low-abundance components, as there is less compromise with detector response than in reduction to the +1 charge state. Preliminary evidence also suggests that the ion collision cross section may play an important role in determining which charge states are most significantly inhibited from further ion/ion reactions under a given set of ion parking conditions.     

Detection of native protein ions in aqueous solution under ambient conditions by electrospray laser desorption/ionization mass spectrometry

Liquid electrospray laser desorption/ionization (ELDI) mass spectrometry allows desorption and ionization of proteins directly from aqueous solutions and biological fluids under ambient conditions. Native protein ions such as those of myoglobin, cytochrome c, and hemoglobin were obtained. A droplet (ca. 5 microL) containing the protein molecules and micrometer-sized particles (e.g., carbon graphite powder) is irradiated with a pulsed UV laser. The laser energy adsorbed by the inert particles is transferred to the surrounding solvent and protein molecules, leading to their desorption; the desorbed gaseous molecules are then postionized within an electrospray (ESI) plume to generate the ESI-like protein ions. With the use of this technique, we detected only the protonated protein ions in various biological fluids (including human tears, cow milk, serum, and bacterial extracts) without interference from their corresponding sodiated or potassiated adduct ions. In addition, we rapidly quantified the levels of glycosylated hemoglobin present in drops of whole blood obtained from diabetic patients without the need of sample pretreatment.     

Mass spectrometry screening of combinatorial mixtures, correlation of measured and predicted electrospray ionization spectra

Methodology was developed to afford rapid characterization of multicomponent mixtures of small organic molecules prepared by split-and-mix combinatorial synthesis. This methodology involved the use of liquid chromatography mass spectrometry (LC/MS) combined with correlation analysis of measured versus predicted electrospray ionization mass spectra. Low-resolution mass spectra of complex mixtures revealed predictable patterns that confirm library products, assisted in identifying chemical synthesis errors, and assessed overall library integrity. In general, equal signal intensities were observed for most combinatorial mixture components, indicating that differences in electrospray ionization efficiency was not a major limitation to this approach. High-throughput data processing programs and informatics tools were used to speed data analysis and to simplify the presentation of the library characterization results. This approach has been used to characterize combinatorial libraries that were synthesized for a variety of drug-discovery programs. Examples are shown for library formats of 1, 40, 66, 280, and 400 component(s)/well. The applicability of this approach to large combinatorial mixtures should allow direct characterization of massive combinatorial libraries.     

Analysis of microbial mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Many different laboratories are currently developing mass-spectrometric techniques to analyze and identify microorganisms. However, minimal work has been done with mixtures of bacteria. To demonstrate that microbial mixtures could be analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), mixed bacterial cultures were analyzed in a double-blind fashion. Nine different bacterial species currently in our MALDI-MS fingerprint library were used to generate 50 different simulated mixed bacterial cultures similar to that done for an initial blind study previously reported (Jarman, K. H.; Cebula, S. T.; Saenz, A. J.; Petersen, C. E.; Valentine, N. B.; Kingsley, M. T.; Wahl, K. L. Anal. Chem. 2000, 72, 1217-1223). The samples were analyzed by MALDI-MS with automated data extraction and analysis algorithms developed in our laboratory. The components present in the sample were identified correctly to the species level in all but one of the samples. However, correctly eliminating closely related organisms was challenging for the current algorithms, especially in differentiating Serratia marcescens, Escherichia coli, and Yersinia enterocolitica, which have some similarities in their MALDI-MS fingerprints. Efforts to improve the specificity of the algorithms are in progress.     

Dissolution of willemite polycrystals: effects of pH,temperature and TiO2 solid solution

Dissolution (in terms of weight loss) experiments on willemite in the form of sintered polycrystals (TiO₂-dissolved or TiO₂-free) were conducted over a wide solution pH range (pH 1–13) at 25 and 50°C, respectively, or over the temperature range 25–90°C at pH 1. Dissolution follows a linear kinetics in acidic or basic solutions; the apparent activation energy of acid dissolution (pH 1) is 19 and 16 kJ mol^–1, respectively, for the TiO₂-free and the TiO₂-dissolved willemite. The pH dependence of the dissolution behaviour resembles that of zinc oxide rather than silica. Willemite polycrystals dissolve via parabolic-like kinetics in the intermediate pH range, which may be attributed to the formation of a passive film or to the possible polishing effect. TiO₂ solid solution facilitates acid but suppresses base dissolution of willemite, but grain boundary dissolution also contributes significantly in the basic region.     

Supercharged protein and peptide ions formed by electrospray ionization

The multiple charging of large molecules in electrospray ionization provides key advantages for obtaining accurate molecular weights by mass spectrometry and for obtaining structural information by tandem mass spectrometry and MS(n) experiments. Addition of glycerol or m-nitrobenzyl alcohol into the electrospray solutions dramatically increases both the maximum observed charge state and the abundances of the high charge states of protein and peptide ions. Adding glycerol to acidified aqueous solutions of cytochrome c shifts the most abundant charge state from 17+ to 21+, shifts the maximum charge state from 20+ to 23+, and shifts the average charge state from 16.6+ to 20.9+. Much less m-nitrobenzyl alcohol (<1%) is required to produce similar results. With just 0.7% m-nitrobenzyl alcohol, even the 24+ charge state of cytochrome c is readily observed. Similar results are obtained with myoglobin and (Lys)4. For the latter molecule, the 5+ charge state is observed in the electrospray mass spectrum obtained from solutions containing 6.7% m-nitrobenzyl alcohol. This charge state corresponds to protonation of all basic sites in this peptide. Although the mechanism for enhanced charging is unclear, it does not appear to be a consequence of conformational changes of the analyte molecules. This method of producing highly charged protein ions should be useful for improving the performance of mass measurements on mass spectrometers with performances that decrease with increasing m/z. This should also be particularly useful for tandem mass spectrometry experiments, such as electron capture dissociation, for which highly charged ions are desired.