Studies on heme binding in myoglobin,hemoglobin, and cytochrome c by ion spray mass spectrometry

The ion spray mass spectra of three representative heme-containing proteins were studied, with an emphasis on results obtained under neutral (pH 7) aqueous conditions. The noncovalently bound heme in myoglobin and hemoglobin may be readily distinguished from the covalently bound heme prosthetic group attached to cytochrome c by using collisioninduced dissociation in the free-jet expansion region of the mass spectrometer as well as in the collision quadrupole with premass selection. The charge state of iron in the expelled heme from myoglobin and hemoglobin appears to be 3+ but 2f for heme expelled from cytochrome c.     

Preservation of non-covalent associations in electrospray ionization mass spectrometry: Multiply charged polypeptide and protein dimers

The ‘softness’ of the electrospray ionization (ESI) method provides a direct link between solution chemistry and the inherent gas-phase environment of mass Spectrometry. Available results related to the preservation of non-covalent associations into the gas phase after ESI are reviewed. These associations include the possible retention of elements of higher order protein structure, non-covalent polypeptide–heme associations and enzyme complexes. Experimental results are presented showing that non-covalently bound polypeptide and protein dimer ions are relatively common as low level contributions to ESI mass spectra. It is argued that these dimers are reflective of multimeric species in solution since Coulombic barriers preclude dimerization after ESI although uncertainty remains regarding whether they exist prior to the formation of highly charged droplets. The dissociation of dimers is facile and for proteins can yield monomers having a broad distribution of charge states. The detection of non-covalently associated dimers requires gentle ESI mass spectrometer interface conditions, yielding relatively low levels of internal excitation. Under such conditions incomplete molecular ion desolvation can result in experimental artifacts for tandem mass spectrometric experiments. ESI mass Spectrometry may have broad potential for the study of noncovalent liquid phase associations.     

Primary to quaternary protein structure determination with electrospray ionization and magnetic sector mass spectrometry

Electrospray ionization with a forward-geometry magnetic sector mass spectrometer was used for collisionally activated dissociation studies of multiply charged polypeptides and for studying non-covalently bound protein systems. The high-resolution capabilities of a high-performance instrument allow the resolution of isotopic contributions for product ions and molecular ion species. Determination of product ion charge states by this method reduces difficulties in the interpretation of product ion mass spectra from multiply charged precursors, which are generated either in the atmospheric pressure/vacuum electrospray interface or in the collision chamber of the mass spectrometer. Extended tandem mass spectrometric experiments have the potential for sequencing larger polypeptides. However, evidence for isomerization of gas-phase product ions from substance P and substance P analogues was observed, complicating the interpretation of product ion spectra. Non-covalent complexes can also be studied by electrospray ionization magnetic sector MS. The higher m/z range of such an instrument is a major advantage for studying weakly bound systems, such as heme–protein systems (myoglobin, hemoglobin) and protein aggregates (concanavalin A), because of their tendency to form complex ions with relatively low charge states.     

The use of tandem mass spectrometry for the differentiation of bile acid isomers and for the identification of bile acids in biological extracts

Tandem mass spectrometric (MS/MS) techniques hae been widely used for the differentiation of isomeric compounds, since their spectra may show differences sufficient to distinguish between them. There are several different ways by which the MS/MS data can be obtained depending on the energies of the ions and the collisions. In this paper MS/MS spectra have been obtained for a group of isomeric bile acids using: 1, low-energy ions and low-energy collisions in a triple quadrupole mass spectrometer by liquid chromatography/MS/MS; 2, high-energy ions and low-energy collisions in a hybrid mass spectrometer by fast-atom bombardment MS/MS. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) has also been used to identify the bile acids present in biological matrices such as bile extracts.     

Mutation in the flavin mononucleotide domain modulates magnetic circular dichroism spectra of the iNOS ferric cyano complex in a substrate-specific manner

We have obtained low-temperature magnetic circular dichroism (MCD) spectra for ferric cyano complexes of the wild type and E546N mutant of a human inducible nitric oxide synthase (iNOS) oxygenase/flavin mononucleotide (oxyFMN) construct. The mutation at the FMN domain has previously been shown to modulate the MCD spectra of the l-arginine-bound ferric iNOS heme (Sempombe, J.; et al. J. Am. Chem. Soc. 2009, 131, 6940-6941). The addition of l-arginine to the wild-type protein causes notable changes in the CN(-)-adduct MCD spectrum, while the E546N mutant spectrum is not perturbed. Moreover, the MCD spectral perturbation observed with l-arginine is absent in the CN(-) complexes incubated with N-hydroxy-L-arginine, which is the substrate for the second step of NOS catalysis. These results indicate that interdomain FMN-heme interactions exert a long-range effect on key heme axial ligand-substrate interactions that determine substrate oxidation pathways of NOS.     

Conformer selection of protein ions by ion mobility in a triple quadrupole mass spectrometer

Electrospray mass spectra of multiply charged protein molecules show two distinct charge state distributions proposed to correspond to a more highly charged, open conformational form and a lower charged, folded form. Elastic collisions carried out in the radiofrequency-only collision cell of a triple quadrupole mass spectrometer have dramatic effects on the appearance of the mass spectra. The different cross sectional areas of the conformers allow preferential selection of one charge state distribution over the other on the basis of ion mobility. Preferential selection is dependent on the nature and pressure of the target gas as well as the nature of the protein. In the case of positively charged horse heart apomyoglobin (MW 16,951 da), a high charge state distribution centered around (M + 20H)²⁰⁺ predominates at low target gas pressures and a second distribution centered around (M + 10H)¹⁰⁺ predominates at high target gas pressures. Bimodal distributions are observed at intermediate pressures and, remarkably, charge states between the two distributions are not effectively populated under most of the conditions examined. Hard sphere collision calculations show large differences in collision frequencies and in the corresponding kinetic energy losses for the two conformational states and they demonstrate that the observed charge state selectivity can be explained through elastic collisions.     

Investigation of collisional-activation decomposition process and spectra in the transport region of an electrospray single-quadrupole mass spectrometer.

The use of collisional-activation dissociation (CAD) in the electrospray transport region was evaluated for generating structural information on several pesticides and antibiotics. The collision energy used to generate the CAD spectra could be varied easily by changing the capillary/skimmer potential difference, imparting from 0 eV to above 16 eV internal energy to the near thermal ions generated by electrospray. The internal energy distribution for low-energy collisions (capillary/skimmer potential difference of 20 V) closely matches the curves generated by a triple-quadrupole mass spectrometer. Furthermore, the CAD spectra for selected compounds generated by electrospray in the transport region at a capillary/skimmer potential difference of 30-50 V closely resembled those obtained from the [M + H]+ ion by a triple quadrupole using 30 eV collision energy. The CAD of ions in the transport region resulted in 70% to 80% daughter-ion yields and minimal loss in overall ion current compared to the ion current for protonated or cationized parent molecules. The major daughter ions for 10 pg of Aldicarb and penicillin G could be detected (signal-to-noise ratio greater than 5) under full-scan CAD conditions.     

Energy-resolved collisional activation of dimethyl phosphonate and dimethyl phosphite ions in a quadrupole ion trap and a triple quadrupole mass spectrometer

Collision-activated dissociation spectra of dimethyl phosphonate and dimethyl phosphite ions were measured as a function of the amplitude of a supplementary AC voltage applied across the end-caps of an ion-trap mass spectrometer. These spectra yield breakdown graphs which bear a close resemblance to those obtained by varying collision energy in a triple-quadrupole mass spectrometer operating under multiple-collision conditions. Variation in the time of excitation at the resonance frequency provides an alternative route to breakdown graphs. The results demonstrate that energy deposition occurs via multiple activating collisions in the ion trap. Maximum energy deposition observed is somewhat smaller under normal operating conditions in the ion trap than in the triple-quadrupole mass spectrometer.     

Characterization of conformational changes and noncovalent complexes of myoglobin by electrospray ionization mass spectrometry,circular dichroism and fluorescence spectroscopy

Electrospray ionization mass spectrometry (ESI‐MS) was employed to monitor the heme release and the conformational changes of myoglobin (Mb) under different solvent conditions, and to observe ligand bindings of Mb. ESI‐MS, complemented by circular dichroism and fluorescence spectroscopy, was used to study the mechanism of acid‐ and organic solvent‐induced denaturation by probing the changes in the secondary and the tertiary structure of Mb. The results obtained show that complete disruption of the heme–protein interactions occurs when Mb is subjected to one of the following solution conditions: pH 3.2–3.6, or solution containing 20–30% acetonitrile or 40–50% methanol. Outside these ranges, Mb is present entirely in its native state (binding with a heme group) or as apomyoglobin (i.e. without the heme). Spectroscopic data demonstrate that the denaturation mechanism of Mb induced by acid may be significantly different from that by the organic solvent. Low pH reduces helices in Mb, whereas certain organic content level in solution results in the loss of the tertiary structure. ESI‐MS conditions were established to observe the H₂O‐ and CO‐bound Mb complexes, respectively. H₂O binding to metmyoglobin (17 585 Da), where the heme iron is in the ferric oxidation state, is observed in ESI‐MS. CO binding to Mb (17 595 Da), on the other hand, can be only observed after the heme iron is reduced to the ferrous form. Therefore, ESI‐MS combined with spectroscopic techniques provides a useful means for probing the formation of ligand‐binding complexes and characterizing protein conformational changes. Copyright © 2010 John Wiley & Sons, Ltd.     

Energy deposition in iron pentacarbonyl ions undergoing surface-induced dissociation in a Fourier transform mass spectrometer

Internal energy deposition into iron pentacarbonyl positive ions undergoing surface-induced dissociation (SID) in a Fourier transform mass spectrometer is estimated from the abundances and known critical energies of the product fragment ions. A narrow energy distribution, comparable to that reported in earlier BQ and tandem quadrupole SID studies of the same compound, is observed. As judged by the ratio of fragment ions to incident parent ions observed, SID of iron pentacarbonyl in the 3 T Fourier transform mass spectrometer is more efficient, but results in lower conversion of laboratory to internal energy. This may be a result of the more shallow collision incidence angle employed in the Fourier transform mass spectrometer measurements (a few degrees), which contrasts with the 32–60° collision angles used in the earlier BQ and tandem quadrupole mass spectrometry studies. Collision-induced dissociation with He under single collision conditions is also reported, Not unexpectedly, conversion of kinetic to internal energy was lower than found in a previous Fourier transform mass spectrometer study of the iron pentacarbonyl cation employing argon as collision gas under multiple collision conditions.     

L-arginine binding to human inducible nitric oxide synthase: an antisymmetric funnel route toward isoform-specific inhibitors?

Nitric oxide (NO) is an important signaling molecule produced by a family of enzymes called nitric oxide synthases (NOS). Because NO is involved in various pathological conditions, the development of potent and isoform-selective NOS inhibitors is an important challenge. In the present study, the dimer of oxygenase domain of human iNOS (iNOSoxy) complexed to its natural substrate L-arginine (L-Arg) and both heme and tetrahydro-L-biopterin (BH4) cofactors was studied through multiple molecular dynamics simulations. Starting from the X-ray structure available for that complex (PDB: 1NSI ), a 16 ns equilibration trajectory was first obtained. Twelve dynamics of slow extraction of L-Arg out from the iNOSoxy active site were then performed. The steered molecular dynamics (SMD) approach was used starting from three different points of the reference trajectory for a total simulation time of 35 ns. A probable unbinding/binding pathway of L-Arg was characterized. It was suggested that a driving force directed the substrate toward the heme pocket. Key intermediate steps/residues along the access route to the active site were identified along this "funnel shape" pathway and compared to existing data. A quasi-normal mode analysis performed on the SMD data suggested that large collective motions of the protein may be involved in L-Arg binding and that opening the route to the active site in one monomer promoted an inverse, closing motion in the second monomer. Finally, our findings might help to rationalize the design of human iNOS isoform competitive inhibitors.     

Direct measurement by laser flash photolysis of intramolecular electron transfer in a two-domain construct of murine inducible nitric oxide synthase

Intersubunit intramolecular electron transfer (IET) from FMN to heme is essential in the delivery of electrons required for O2 activation in the heme domain and the subsequent nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation that serves as the input state for reduction of FMN by electrons from NADPH and FAD in the reductase domain. To favor formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of the IET between the FMN and heme domains in this construct was directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme oxygenase constructs.     

Combined Chemical Modification and Collision Induced Unfolding Using Native Ion Mobility-Mass Spectrometry Provides Insights into Protein Gas-Phase Structure

Native mass spectrometry is now an important tool in structural biology. Thus, the nature of higher protein structure in the vacuum of the mass spectrometer is an area of significant interest. One of the major goals in the study of gas-phase protein structure is to elucidate the stabilising role of interactions at the level of individual amino acid residues. A strategy combining protein chemical modification together with collision induced unfolding (CIU) was developed and employed to probe the structure of compact protein ions produced by native electrospray ionisation. Tractable chemical modification was used to alter the properties of amino acid residues, and ion mobility-mass spectrometry (IM-MS) utilised to monitor the extent of unfolding as a function of modification. From these data the importance of specific intramolecular interactions for the stability of compact gas-phase protein structure can be inferred. Using this approach, and aided by molecular dynamics simulations, an important stabilising interaction between K6 and H68 in the protein ubiquitin was identified, as was a contact between the N-terminus and E22 in a ubiquitin binding protein UBA2.     

Study of the major fragmentation pathways of cypermethrin and related synthetic insecticides using tandem mass spectrometry

Fragmentation pathways of the synthetic pyrethroid cypermethrin and four structurally related insecticides were investigated using a tandem quadrupole mass spectrometer incorporating a hexapole collision cell under positive-ion electron impact ionization conditions. Conventional mass spectrometry using the first quadrupole analyser only and tandem mass spectrometry on selected precursor ions and product ions, and also constant neutral loss scan experiments, were used. Mechanisms and fragmentation pathways are proposed to explain the inherent stability of ions associated with the benzylphenoxy portion of this class of insecticide.     

A study of resonance electron capture ionization on a quadrupole tandem mass spectrometer

Procedures that allow the realization of resonance electron capture (REC) mode on a commercial triple-quadrupole mass spectrometer, after some simple modifications, are described. REC mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments were performed and spectra for some compounds were recorded. In particular, the charge-remote fragmentation (CRF) spectra of [M - H](-) ions of docosanoic and docosenoic acids under low-energy collisionally activated dissociation (CAD) conditions were obtained, and showed that there were no significant differences for [M - H](-) ions produced at different resonances (i.e. for [M - H](-) ions with different structures). This observation was explained on the basis of results obtained from deuterium-labeled fatty acids, which showed that different CRF ions (but with the same m/z value in the absence of labels) could be produced by different mechanisms, and all of them were obviously realized under CAD conditions that made spectra practically indistinguishable. The other example, which compared the REC-MS/MS spectrum of [M - H](-) ions and EI-MS/MS spectrum of M(+.) ions of daidzein, demonstrated the potential of the REC-MS/MS technique for more complex structure elucidation.     

A snapshot of enzyme catalysis using electrospray ionization mass spectrometry

Insights into the early molecular events involving protein-ligand/substrate interactions such as protein signaling and enzyme catalysis can be obtained by examining these processes on a very short, millisecond time scale. We have used time-resolved electrospray mass spectrometry to delineate the catalytic mechanism of a key enzyme in bacterial lipopolysaccharide biosynthesis, 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS). Direct real-time monitoring of the catalytic reaction under single enzyme turnover conditions reveals a novel hemiketal phosphate intermediate bound to the enzyme in a noncovalent complex that establishes the reaction pathway. This study illustrates the successful application of mass spectrometry to reveal transient biochemical processes and opens a new time domain that can provide detailed structural information of short-lived protein-ligand complexes.     

Mechanism of inactivation of inducible nitric oxide synthase by amidines. Irreversible enzyme inactivation without inactivator modification

Nitric oxide synthases (NOS) are hemoproteins that catalyze the reaction of L-arginine to L-citrulline and nitric oxide. N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was reported to be a slow, tight-binding, and highly selective inhibitor of iNOS in vitro and in vivo. Previous mechanistic studies reported that 1400W was recovered quantitatively after iNOS fully lost its activity and modification to iNOS was not detected. Here, it is shown that 1400W is a time-, concentration-, and NADPH-dependent irreversible inactivator of iNOS. HPLC-electrospray mass spectrometric analysis of the incubation mixture of iNOS with 1400W shows both loss of heme cofactor and formation of biliverdin, as was previously observed for iNOS inactivation by another amidine-containing compound, N5-(1-iminoethyl)-L-ornithine (L-NIO). The amount of biliverdin produced corresponds to the amount of heme lost by 1400W inactivation of iNOS. A convenient MS/MS-HPLC methodology was developed to identify the trace amount of biliverdin produced by inactivation of iNOS with either 1400W or L-NIO to be biliverdin IXalpha out of the four possible regioisomers. Two mechanisms were previously proposed for iNOS inactivation by L-NIO: (1) uncoupling of the heme peroxide intermediate, leading to destruction of the heme to biliverdin; (2) abstraction of a hydrogen atom from the amidine methyl group followed by attachment to the heme cofactor, which causes the enzyme to catalyze the heme oxygenase reaction. The second mechanistic proposal was ruled out by inactivation of iNOS with d3-1400W, which produced no d2-1400W. Detection of carbon monoxide as one of the heme-degradation products further excludes the covalent heme adduct mechanism. On the basis of these results, a third mechanism is proposed in which the amidine inactivators of iNOS bind as does substrate L-arginine, but because of the amidine methyl group, the heme peroxy intermediate cannot be protonated, thereby preventing its conversion to the heme oxo intermediate. This leads to a change in the enzyme mechanism to one that resembles that of heme oxygenase, an enzyme known to convert heme to biliverdin IXalpha. This appears to be the first example of a compound that causes irreversible inactivation of an enzyme without itself becoming modified in any way.     

A glycomics platform for the analysis of permethylated oligosaccharide alditols

This communication reports the development of an LC/MS platform for the analysis of permethylated oligosaccharide alditols that, for the first time, demonstrates routine online oligosaccharide isomer separation of these compounds before introduction into the mass spectrometer. The method leverages a high-resolution liquid chromatography system with the superior fragmentation pattern characteristics of permethylated oligosaccharide alditols that are dissociated under low-energy collision conditions using quadrupole orthogonal time-of-flight (QoTOF) instrumentation and up to pseudo MS(3) mass spectrometry. Glycoforms, including isomers, are readily identified and their structures assigned. The isomer-specific spectra include highly informative cross-ring and elimination fragments, branch position specific signatures, and glycosidic bond fragments, thus facilitating linkage, branch, and sequence assignment. The method is sensitive and can be applied using as little as 40 fmol of derivatized oligosaccharide. Because permethylation renders oligosaccharides nearly chemically equivalent in the mass spectrometer, the method is semiquantitative and, in this regard, is comparable to methods reported using high field NMR and capillary electrophoresis. In this postgenomic age, the importance of glycosylation in biological processes has become clear. The nature of many of the important questions in glycomics is such that sample material is often extremely limited, thus necessitating the development of highly sensitive methods for rigorous structural assignment of the oligosaccharides in complex mixtures. The glycomics platform presented here fulfills these criteria and should lead to more facile glycomics analyses.     

A model for energy transfer in inelastic molecular collisions applicable at steady state or non-steady state and for an arbitrary distribution of collision energies

A new model for energy exchange between translational and internal degrees of freedom in atom-molecule collisions has been developed. It is suitable for both steady state conditions (e.g., a large number of collisions with thermal kinetic energies) and non-steady state conditions with an arbitrary distribution of collision energies (e.g., single high-energy collisions). In particular, it does not require that the collision energies be characterized by a quasi-thermal distribution, but nevertheless it is capable of producing a Boltzmann distribution of internal energies with the correct internal temperature under quasi-thermal conditions. The energy exchange is described by a transfer probability density that depends on the initial relative kinetic energy, the internal energy of the molecule, and the amount of energy transferred. The probability density for collisions that lead to excitation is assumed to decrease exponentially with the amount of transferred energy. The probability density for de-excitation is obtained from microscopic reversibility. The model has been implemented in the ion trap simulation program ITSIM and coupled with an Rice-Rampsberger-Kassel-Marcus (RRKM) algorithm to describe the unimolecular dissociation of populations of ions. Monte Carlo simulations of collisional energy transfer are presented. The model is validated for non-steady state conditions and for steady state conditions, and the effect of the kinetic energy dependence of the collision cross-section on internal temperature is discussed. Applications of the model to the problem of chemical mass shifts in RF ion trap mass spectrometry are shown.     

An electrospray-ionization mass spectrometry analysis of the pH-dependent dissociation and denaturation processes of a heterodimeric protein

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analysis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associate under acidic conditions, but become dissociated and denatured under neutral and basic conditions. In order to understand the unique pH-dependent denaturation mechanism of this protein, a pH titration was performed by utilizing ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subunits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-dependent dissociation occurs in the liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of this protein. In conclusion, the pH titration of a protein by ESI-MS is particularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules.