Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes

Electrospray ionization (ESI) mass spectrometry (MS) in both the positive and negative ion mode has been used to study protein unfolding transitions of lysozyme, cytochrome c (cyt c), and ubiquitin in solution. As expected, ESI of unfolded lysozyme leads to the formation of substantially higher charge states than the tightly folded protein in both modes of operation. Surprisingly, the acid-induced unfolding of cyt c as well as the acid and the base-induced unfolding of ubiquitin show different behavior: In these three cases protein unfolding only leads to marginal changes in the negative ion charge state distributions, whereas in the positive ion mode pronounced shifts to higher charge states are observed. This shows that ESI MS in the negative ion mode as a method for probing conformational changes of proteins in solution should be treated with caution. The data presented in this work provide further evidence that the conformation of a protein in solution not its charge state is the predominant factor for determining the ESI charge state distribution in the positive ion mode. Furthermore, these data support the hypothesis of a recent study (Konermann and Douglas, Biochemistry 1997, 36, 12296–12302) which suggested that ESI in the positive ion mode is not sensitive to changes in the secondary structure of proteins but only to changes in the tertiary structure.     

Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI,nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry

We present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of standard electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to determine the dissociation constant (KD) of the model system hen egg white lysozyme (HEWL) binding to N,N',N'-triacetylchitotriose (NAG3). The best KD value compared with solution data were found for ESSI, 19.4 +/- 3.6 microM. Then, we determined the KDs of the two nucleotide binding sites of adenylate kinase (AK), where we obtained KDs of 2.2 +/- 0.8 microM for the first and 19.5 +/- 8.0 microM for the second binding site using ESSI. We found a weak charge state dependence of the KD for both protein-ligand systems, where for all ionization techniques the KD value decreases with increasing charge state. We demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the KD obtained is in good agreement with solution phase results from the literature. In addition, we tried to determine the KD for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amount of sample available. In this case, we found KD values with a strong charge state dependence, which were in no case close to literature values for solution phase.     

Top-down ESI-ECD-FT-ICR mass spectrometry localizes noncovalent protein-ligand binding sites

Mass spectrometry (MS) with electrospray ionization (ESI) has the capability to measure and detect noncovalent protein-ligand and protein-protein complexes. However, information on the sites of ligand binding is not easily obtained by the ESI-MS methodology. Electron capture dissociation (ECD) favors cleavage of covalent backbone bonds of protein molecules. We show that this characteristic of ECD translates to noncovalent protein-ligand complexes, as covalent backbone bonds of protein complexes are dissociated, but the noncovalent ligand interaction is retained. For the complex formed from 140-residue, 14.5 kDa alpha-synuclein protein, and one molecule of polycationic spermine (202 Da), ECD generates product ions that retain the protein-spermine noncovalent interaction. Spermine binding is localized to residues 106-138; the ECD data are consistent with previous solution NMR studies. Our studies suggest that ECD mass spectrometry can be used to determine directly the sites of ligand binding to protein targets.     

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

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.     

Study of the noncovalent interactions of ginsenosides and amyloid‐β‐peptide by CSI‐MS and molecular docking

Noncovalent interactions between drugs and proteins play significant roles for drug metabolisms and drug discoveries. Mass spectrometry has been a commonly used method for studying noncovalent interactions. However, the harsh ionization process in electrospray ionization mass spectrometry (ESI‐MS) is not conducive to the preservation of noncovalent and unstable biomolecular complexes compared with the cold spray ionization mass spectrometry (CSI‐MS). A cold spray ionization providing a stable solvation‐ionization at low temperature is milder than ESI, which was more suitable for studying noncovalent drug‐protein complexes with exact stoichiometries. In this paper, we apply CSI‐MS to explore the interactions of ginsenosides toward amyloid‐β‐peptide (Aβ) and clarify the therapeutic effect of ginsenosides on Alzheimer's disease (AD) at the molecular level for the first time. The interactions of ginsenosides with Aβ were performed by CSI‐MS and ESI‐MS, respectively. The ginsenosides Rg1 bounded to Aβ at the stoichiometries of 1:1 to 5:1 could be characterized by CSI‐MS, while dehydration products are more readily available by ESI‐MS. The binding force depends on the number of glycosyls and the type of ginsenosides. The relative binding affinities were sorted in order as follows: Rg1 ≈ Re > Rd ≈ Rg2 > Rh2, protopanaxatriol by competition experiments, which were supported by molecular docking experiment. CSI‐MS is expected to be a more appropriate approach to determine the weak but specific interactions of proteins with other natural products especially polyhydroxy compounds.     

Application of electrospray mass spectrometry in probing protein-protein and protein-ligand noncovalent interactions

A novel mass spectrometry-based methodology using electrospray ionization (ESI) is described for the detection of protein-protein [interferon (IFN)-γ dimer] and protein-ligand [ras-guanosine diphosphate (GDP)] noncovalent interactions. The method utilizes ESI from aqueous solution at appropriate pH. The presence of the noncovalent complex of the IFN-γ dimer was confirmed by the observed average molecular weight of 33,819 Da. The key to the detection of the IFN-γ dimer is the use of an alkaline solution (pH ≈ 9) for sample preparation and for mass spectrornetry analysis. The effect of the declustering energy in the region of the ion sampling orifice and focusing quadrupole on the preservation of the gas-phase noncovalent complex (IFN-γ dimer) was also studied. The effect of the declustering energy on complex dissociation was further extended to probe the noncovalent protein-ligand association of ras-GDP. It was found that little energy is required to dissociate the IFN-γ dimer, whereas a substantial amount of energy is required to dissociate the gas-phase ras-GDP complex.     

Cold-spray ionization mass spectrometry: principle and applications

A direct solution analysis method, cold-spray ionization (CSI) mass spectrometry (MS), a variant of electrospray (ESI) MS operating at low temperature (ca -80 to 10 degrees C), allows the facile and precise characterization of labile organic species, especially those in which non-covalent bonding interactions are prominent. We applied this method to investigations of the solution structures of many labile organic species, including unstable reagents and reaction intermediates, asymmetric catalysts, supramolecules and even primary biomolecules. Remarkable analytical results were obtained for highly ordered supramolecules using the CSI method. Whereas conventional ESI is not applicable to these compounds because of their instability to heat and/or air, CSI affords multiply charged molecular ions with many solvent molecules attached. Investigation of the constitution of Grignard reagents in solution is extremely challenging, but CSI-MS allowed us to identify one of the key structures in THF solution. Recently, this method was adopted for investigations of the solution structures of primary biomolecules such as nucleosides, amino acids, sugars and lipids, revealing singly charged Na(+) adducts of large clusters (chain structures), presumably linked by non-covalent interactions, including hydrogen bonding and/or hydrophobic interactions. The principle of the CSI method and applications of the method to a wide variety of labile organic species and primary biomolecules in solution are described.     

Applications of mass spectrometry to food proteins and peptides

The application of mass spectrometry (MS) to large biomolecules has been revolutionized in the past decade with the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques. ESI and MALDI permit solvent evaporation and sublimation of large biomolecules into the gaseous phase, respectively. The coupling of ESI or MALDI to an appropriate mass spectrometer has allowed the determination of accurate molecular mass and the detection of chemical modification at high sensitivity (picomole to femtomole). The interface of mass spectrometry hardware with computers and new extended mass spectrometric methods has resulted in the use of MS for protein sequencing, post-translational modifications, protein conformations (native, denatured, folding intermediates), protein folding/unfolding, and protein-protein or protein-ligand interactions. In this review, applications of MS, particularly ESI-MS and MALDI time-of-flight MS, to food proteins and peptides are described.     

Effect of protein stabilization on charge state distribution in positive- and negative-ion electrospray ionization mass spectra

Changes in protein conformation are thought to alter charge state distributions observed in electrospray ionization mass spectra (ESI-MS) of proteins. In most cases, this has been demonstrated by unfolding proteins through acidification of the solution. This methodology changes the properties of the solvent so that changes in the ESI-MS charge envelopes from conformational changes are difficult to separate from the effects of changing solvent on the ionization process. A novel strategy is presented enabling comparison of ESI mass spectra of a folded and partially unfolded protein of the same amino acid sequence subjected to the same experimental protocols and conditions. The N-terminal domain of the Escherichia coli DnaB protein was cyclized by in vivo formation of an amide bond between its N- and C-termini. The properties of this stabilized protein were compared with its linear counterpart. When the linear form was unfolded by decreasing pH, a charge envelope at lower m/z appeared consistent with the presence of a population of unfolded protein. This was observed in both positive-ion and negative-ion ESI mass spectra. Under the same conditions, this low m/z envelope was not present in the ESI mass spectrum of the stable cyclized form. The effects of changing the desolvation temperature in the ionization source of the Q-TOF mass spectrometer were also investigated. Increasing the desolvation temperature had little effect on positive-ion ESI mass spectra, but in negative-ion spectra, a charge envelope at lower m/z appeared, consistent with an increase in the abundance of unfolded protein molecules.     

Detection of noncovalent complex between alpha-amylase and its microbial inhibitor tendamistat by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for detection of noncovalent complexes. However, detection of noncovalent protein-protein complexes is not a widespread practice and still produces some challenges for mass spectrometrists. Here we demonstrate the detection of a noncovalent protein-protein complex between alpha-amylase and its microbial inhibitor tendamistat using ESI-MS. Crude porcine pancreatic alpha-amylase was purified using a glycogen precipitation method. Noncovalent complexes between porcine pancreatic alpha-amylase and its microbial inhibitor tendamistat are probed and detected using ESI-MS. The atmosphere-vacuum ESI conditions along with solution conditions and the ratio of inhibitor over enzyme strongly affect the detection of noncovalent complexes in the gas phase. ESI mass spectra of alpha-amylase at pH 7 exhibited charge states significantly lower than that reported previously, which is indicative of a native protein conformation necessary to produce a noncovalent complex. Detection of noncovalent complexes in the gas phase suggests that further use of conventional biochemical approaches to provide a qualitative, and in some cases even quantitative, characterization of equilibria of noncovalent complexes in solution is possible.     

Effects of pH on the kinetic reaction

In most cases, kinetic unfolding reactions of proteins follow a simple one-step mechanism that does not involve any detectable intermediates. One example for a more complicated unfolding reaction is the acid-induced denaturation of holo-myoglobin (hMb). This reaction proceeds through a transient intermediate and can be described by a sequential two-step mechanism (Konermann et al. Biochemistry 1997, 36, 6448-6454). Time-resolved electrospray ionization mass spectrometry (ESI MS) is a new technique for monitoring the kinetics of protein folding and unfolding in solution. Different protein conformations can be distinguished by the different charge state distributions that they generate during ESI. At the same time this technique allows monitoring the loss or binding of noncovalent protein ligands. In this work, time-resolved ESI MS is used to study the dependence of the kinetic unfolding mechanism of hMb on the specific solvent conditions used in the experiment. It is shown that hMb unfolds through a short-lived intermediate only at acidic pH. Under basic conditions no intermediate is observed. These findings are confirmed by the results of optical stopped-flow absorption experiments. This appears to be the first time that a dependence of the kinetic mechanism for protein unfolding on external conditions such as pH has been observed.     

Measurement of inclusion complex formation between cyclophane and biological relevant amino acids using electrospray ionization, cold-spray ionization and fast atom bombardment mass spectrometry

The investigation of the host-guest complex formations between cyclophane (TGDMAP) (1) as a host and L-acidic amino acids such as L-glutamic acid (Glu) and L-aspartic acid (Asp) as guests was carried out using fast atom bombardment (FAB), electrospray ionization (ESI) and cold-spray ionization (CSI) mass spectrometry (MS). The stability constant (K(s)) values obtained by the three different MS methods almost agreed. However, the complex ion peaks of a novel cyclophane (CPCn) (2) with Glu and Asp were not observed in FAB-MS. Then, these host-guest complex formations by use of CSI-MS and ESI-MS was examined, as the results, these complex ion peaks were observed clearly and the measurement values by the two MS methods are mostly in agreement. It was concluded that ESI-MS and CSI-MS are available for the determination of K(s) value as well as FAB-MS.     

Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (Ω) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra- and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding.     

Critical evaluation of mass spectrometric measurement of dissociation constants: accuracy and cross-validation against surface plasmon resonance and circular dichroism for the calmodulin-melittin system

We present a comprehensive study for determining the binding affinity of a protein-ligand complex, using mass spectrometric methods. Mass spectrometry has been used to study noncovalent interactions for a number of years. However, the use of soft ionization mass spectrometry for quantitative analysis of noncovalently bound complexes is not widely accepted. This paper reports a comparison of MS methods against established methods such as surface plasmon resonance (SPR) and circular dichroism (CD) whose suitability for the quantitative assessment of noncovalent interactions is well known. ESI titration and MALDI-SUPREX were used as representative mass spectrometric methods for this work. We chose to study the calmodulin-melittin complex that presents three challenges: (i) it exhibits a high affinity (low nanomolar KD); (ii) complexes are formed only in the presence of a coactivator, calcium ions in this case; and (iii) the protein and the complex show a different ionization efficiency. Dissociation constants were obtained from each method for the selected system and compared thoroughly to elucidate pros and cons of the selected methodologies in terms of their ability for the determination of binding constants of protein-ligand complexes. ESI titration, SPR, CD and MALDI-SUPREX yielded KD values in the low nanomolar range that are in general agreement with an older value reported in the literature. We also critically evaluated the limitations in particular of the MS methods and the associated data evaluation procedures. We present an improved evaluation of SUPREX data, as well as a detailed error analysis for all methods used.     

The study of protein conformation in solution via direct sampling by desorption electrospray ionization mass spectrometry

The direct sampling feature of liquid sample desorption electrospray ionization (DESI) allows the ionization of liquid samples without adding acids/organic solvents (i.e., without sample pretreatment). As a result, it provides a new approach for probing protein conformation in solution. In this study, it has been observed that native protein ions are generated from proteins in water by DESI. Interestingly, the intensities of the resulting protein ions appear to be higher than those generated by ESI of the proteins in water or in ammonium acetate. For protein solutions that already contain acids/organic solvents, DESI can be used to investigate the influences of these denaturants on protein conformations and the obtained results are in good agreement with spectroscopic data. In addition, online monitoring of protein conformational changes by DESI is feasible; for instance, heat-induced unfolding of ubiquitin can be traced with DESI in water without influences of organic solvents/acids. This DESI method provides a new alternative tool for the study of protein conformation in solution.     

Electrospray mass spectra from protein electroeluted from sodium dodecylsulfate polyacrylamide gel electrophoresis gels

Electrospray ionization/tandem mass spectrometry of proteins separated on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels is severely limited by the requirement that the protein be completely separated from the SDS. As shown here, the gaseous noncovalent SDS adducts of protonated proteins thus formed can be dissociated by infrared irradiation. ESI spectra from myoglobin in SDS-containing solutions show molecular ions adducted with up to 15 dodecyl sulfates, but ir irradiation of these ions causes complete dissociation to expel the SDS.     

Monitoring the Tanford transition in β‐lactoglobulin by 8‐anilino‐1‐naphthalene sulfonate and mass spectrometry

The fluorescent dye 8‐anilino‐1‐naphthalene sulfonate (ANS) is known to interact with proteins by conformation‐specific hydrophobic interactions and rather nonspecific electrostatic interactions. To which category the complexes detectable by mass spectrometry (MS) belong is still the subject of debate. Here, the Tanford transition in β‐lactoglobulin (BLG) is exploited as an experimental device to expose hydrophobic binding sites by an increase in pH, rather than, as usually done, by lowering the pH. Complex formation is monitored by electrospray ionization (ESI)‐MS and fluorescence spectroscopy. Both techniques reveal stronger ANS binding to BLG at pH 7.9 than at pH 5.9, suggesting that dye binding inside the calyx, which is known to be hydrophobically driven in solution, can contribute to the complexes detected by ESI‐MS. Electrostatic interactions between the protein and the ANS sulfonate group can only be weaker at pH 7.9 than at pH 5.9, supporting the interpretation of the results by the protein conformational change. Lysozyme is used as a negative control, which shows no variation in the interaction with ANS in the same range of pH, in the absence of conformational changes. However, comparison of MS and fluorescence data at variable pH for BLG and myoglobin (Mb) suggests that conformation‐specific ANS binding to proteins is detectable by ESI‐MS only inside well‐structured cavities of folded structures, like the BLG calyx and apoMb heme pocket. Indeed, ANS interactions with highly dynamic structures or molten globules, although detectable in solution, are easily lost in the gas phase. Copyright © 2008 John Wiley & Sons, Ltd.     

Direct monitoring of protein-chemical reactions utilising nanoelectrospray mass spectrometry

The feasibility of nanoelectrospray mass spectrometry (nanoESI) for the direct analysis of protein chemical reactions and structural changes of proteins has been evaluated. Taking advantage of the long spraying time and the capability of nanoESI for employing a wide range of solvent conditions such as buffers and detergents, applications of monitoring reaction pathways, and dynamics have been carried out with several peptides and proteins. The time course of proteolytic digestions with trypsin and pepsin was investigated for several model polypeptides, and nanoESI showed to provide an efficient tool for optimising digestion conditions for the mass spectrometric peptide mapping analysis. Examples of specific protein chemical modification reactions at arginine and tyrosine residues illustrate the feasibility of nanoESI to monitoring reaction yields and modification sites for more than 180 min. Furthermore, changes of the pattern of protonated molecules caused by temperature effects and by protein unfolding due to disulfide bond reduction have been studied with the model proteins cytochrome c and hen eggwhite lysozyme. The results indicate that nanoESI is an efficient technique for the direct, molecular characterisation of protein-chemical reactions in solution.     

Effect of solution acidity on cytochrome c conformations of alternating current electrospray ionization mass spectrometry

This paper reports notable observations regarding the ion charge states of thermally stable cytochrome c, generated using an alternating current (AC) electrospray ionization (ESI) device. An AC ESI sprayer entrains low-mobility ions to accumulate at the meniscus cone tip prior to the ejection of detached aerosols to produce analyte ions. Therefore, as the solvent acidity varies, protein ions entrained in the AC cone tip are found to change conformation less significantly compared with those in the direct current (DC) cone. We acquired the AC ESI mass spectra of cytochrome c at pH range from 2 to 4. Unlike the DC ESI mass spectra showing clear conformation changes due to denaturing, the AC spectra indicated that only partial denaturing occurs even at extremely acidic pH 2. More native cytochrome c in lower charge states therefore remained. Moreover, with a solvent mixture of aqueous buffer and acetonitrile (70:30), partially denatured cytochrome c was still preserved at pH 2 by using AC ESI. Completely denatured proteins are observed at pH 2 by using DC ESI.     

Evidence for the Preservation of Native Inter- and Intra-Molecular Hydrogen Bonds in the Desolvated FK-Binding Protein·FK506 Complex Produced by Electrospray Ionization

It is now well established that electrospray ionization (ESI) is capable of introducing noncovalent protein assemblies into a desolvated environment, thereby allowing their analysis by mass spectrometry. The degree to which native interactions from the solution phase are preserved in this environment is less clear. Site-directed mutagenesis of FK506-binding protein (FKBP) has been employed to probe specific intra- and inter-molecular interactions within the complex between FKBP and its ligand FK506. Collisional activation of wild-type and mutant-FKBP?FK506 ions, generated by ESI, demonstrated that removal of native protein-ligand interactions formed between residues Asp37, Tyr82, and FK506 significantly destabilized the complex. Mutation of Arg42 to Ala42, or Tyr26 to Phe26 also resulted in lower energy dissociation of the FKBP·FK506 complex. Although these residues do not form direct H-bonds to FK506, they interact with Asp37, ensuring its correct orientation to associate with the ligand. Comparison with solution-based affinity measurements of these mutants has been discussed, including the stabilization afforded by ordered water molecules. Ion mobility spectrometry (IMS) has been employed to provide gas-phase structural information on the unfolding of the complexes. The [M + 6H](6+) complexes of the wild-type and mutants have been shown to resist unfolding and retain compact conformations. However, removal of the basic Arg42 residue was found to induce significant structural weakening of the [M + 7H](7+) complex when raised to dissociation-level energies. Overall, destabilization of the FKBP·FK506 complex, resulting from targeted removal of specific H-bonds, provides evidence for the preservation of these interactions in the desolvated wild-type complex.