Dissociation of heme from gaseous myoglobin ions studied by infrared multiphoton dissociation spectroscopy and Fourier-transform ion cyclotron resonance mass spectrometry

Detachment of heme prosthetic groups from gaseous myoglobin ions has been studied by collision-induced dissociation and infrared multiphoton dissociation in combination with Fourier-transform ion cyclotron resonance mass spectrometry. Multiply charged holomyoglobin ions (hMbn+) were generated by electrospray ionization and transferred to an ion cyclotron resonance cell, where the ions of interest were isolated and fragmented by either collision with Ar atoms or irradiation with 3 mum photons, producing apomyoglobin ions (aMbn+). Both charged heme loss (with [Fe(III)-heme]+ and aMb(n-1)+ as the products) and neutral heme loss (with [Fe(II)-heme] and aMbn+ as the products) were detected concurrently for hMbn+ produced from a myoglobin solution pretreated with reducing reagents. By reference to Ea = 0.9 eV determined by blackbody infrared radiative dissociation for charged heme loss of ferric hMbn+, an activation energy of 1.1 eV was deduced for neutral heme loss of ferrous hMbn+ with n = 9 and 10.     

Coulomb effects in binding of heme in gas-phase ions of myoglobin

Coulomb effects in binding of heme in gas-phase holomyoglobin ions are studied. Positive and negative ions are formed from solution myoglobin with Fe(2+) (ferromyoglobin) and Fe(3+) (ferrimyoglobin). The energy that must be added to the resulting holomyoglobin ions to cause heme loss has been measured by triple-quadrupole tandem mass spectrometry. With negative ions, neutral heme is lost regardless of the charge state of Fe in solution. It is likely that the Fe(3+) is reduced to Fe(2+) in the negative electrospray process. With positive ions, predominantly neutral heme loss is observed with ions formed from ferromyoglobin in solution, and positive heme loss with ions formed from ferrimyoglobin in solution. The energies required to induce neutral heme loss are similar for positive and negative ions. The energies required to induce charged heme loss from positive holomyoglobin ions are significantly less. Coulomb repulsion between the charged heme and charged protein appears to lower the barrier for heme loss. These results are consistent with a simple model potential with a long-range Coulomb repulsion and short-range attraction between the heme and protein.     

Ion Trap Collision-Induced Dissociation of Human Hemoglobin α-Chain Cations

Multiply protonated human hemoglobin alpha-chain species, ranging from [M + 4H]4+ to [M + 20H]20+, have been subjected to ion trap collisional activation. Cleavages at 88 of the 140 peptide bonds were indicated, summed over all charge states, although most product ion signals were concentrated in a significantly smaller number of channels. Consistent with previous whole protein ion dissociation studies conducted under similar conditions, the structural information inherent to a given precursor ion was highly sensitive to charge state. A strongly dominant cleavage at D75/M76, also noted previously in beam-type collisional activation studies, was observed for the [M + 8H]8+ to [M + 11H]11+ precursor ions. At lower charge states, C-terminal aspartic acid cleavages were also prominent but the most abundant products did not arise from the D75/M76 channel. The [M + 12H]12+-[M + 16H]16+ precursor ions generally yielded the greatest variety of amide bond cleavages. With the exception of the [M + 4H]4+ ion, all charge states showed cleavage at the L113/P114 bond. This cleavage proved to be the most prominent dissociation for charge states [M + 14H]14+ and higher. The diversity of dissociation channels observed within the charge state range studied potentially provides the opportunity to localize residues associated with variants via a top-down tandem mass spectrometry approach.     

Ion-ion proton transfer reactions of bio-ions involving noncovalent interactions: Holomyoglobin

Multiply protonated horse skeletal muscle holomyoglobin and apomyoglobin have been subjected to ion-ion proton transfer reactions with anions derived from perfluoro-1,3-dimethylcyclohexane in a quadrupole ion trap operated with helium as a bath gas at 1 mtorr. Neither the apomyoglobin nor holomyoglobin ions show any sign of fragmentation associated with charge state reduction to the 1 + charge state. This is particularly noteworthy for the holomyoglobin ions, which retain the noncovalently bound heme group. For example, no sign of heme loss is associated with charge state reduction from the 9 + charge state of holomyoglobin to the 1 + charge state despite the eight consecutive highly exothermic proton transfer reactions required to bring about this charge change. This result is consistent with calculations that show the combination of long ion lifetime and the high ion-helium collision rate relative to the ion-ion collision rate makes fragmentation unlikely for high mass ions in the ion trap environment even for noncovalently bound complexes of moderate binding strength. The ion-ion proton transfer rates for holo- and apomyoglobin ions of the same charge state also were observed to be indistinguishable, which supports the expectation that ion-ion proton transfer rates are insensitive to ion structure and are determined primarily by the attractive Coulomb field.     

Asymmetric charge partitioning upon dissociation of DNA duplexes

Upon collisional activation, a series of DNA duplexes exhibited a significant degree of asymmetric dissociation with respect to charge partitioning among the single strands. That is, the charge states of the single strand product ions did not equal q/2 for even precursor charge states or (q + 1)/2 and (q − 1)/2 for odd precursor charge states (where q is the charge of the precursor). The factors that affect this asymmetric charge partitioning were assessed. The smaller, lower charged duplexes resulted in more symmetric dissociation compared with larger duplexes in higher charge states, which displayed a high degree of asymmetry upon dissociation. The composition of the duplexes influenced charge partitioning, with those containing a greater number of A/T base pairs showing more symmetric dissociation relative to the more G/C rich duplexes. The use of higher collisional energies resulted in significantly more asymmetric dissociation. Comparisons were made with the dissociation behavior previously studied for protein noncovalent complexes and past studies of the gas-phase conformations and dissociation of DNA complexes.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

The role of conformation on electron capture dissociation of ubiquitin

Effects of protein conformation on electron capture dissociation (ECD) were investigated using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and Fourier-transform ion cyclotron resonance mass spectrometry. Under the conditions of these experiments, the electron capture efficiency of ubiquitin 6+ formed from three different solution compositions differs significantly, ranging from 51 +/- 7% for ions formed from an acidified water/methanol solution to 88 +/- 2% for ions formed from a buffered aqueous solution. This result clearly indicates that these protein ions retain a memory of their solution-phase structure and that conformational differences can be probed in an ECD experiment. Multiple conformers for the 7+ and 8+ charge states of ubiquitin were separated using FAIMS. ECD spectra of conformer selected ions of the same charge states differ both in electron capture efficiency and in the fragment ion intensities. Conformers of a given charge state that have smaller collisional cross sections can have either a larger or smaller electron capture efficiency. A greater electron capture efficiency was observed for ubiquitin 6+ that has the same collisional cross section as one ubiquitin 7+ conformer, despite the lower charge state. These results indicate that the shape of the molecule can have a greater effect on electron capture efficiency than either collisional cross section or charge state alone. The cleavage locations of different conformers of a given charge state were the same indicating that the presence of different conformers in the gas phase is not due to difference in where charges are located, but rather reflect conformational differences most likely originating from solution. Small neutral losses observed from the singly- and doubly-reduced ubiquitin 6+ do not show a temperature dependence to their formation, consistent with these ions being formed by nonergodic processes.     

Factors that Influence Competitive Intermolecular Solvation of Protonated Groups in Peptides and Proteins in the Gas Phase

The ability of 18-crown-6 (18C6) to form noncovalent complexes with cationic groups in the gas phase has been leveraged in numerous, largely orthogonal mass spectrometry-based applications. Although the fundamental interaction between 18C6 and a charged group in the gas phase is quite strong, the strength of attachment of 18C6 to large molecules is more difficult to predict because intramolecular binding of the cation can be competitive. Herein, we demonstrate in experiments with model peptides that 18C6 adducts are not strongly attached to flexible molecules with numerous potential hydrogen bonding sites. 18C6 adduct stability is increased if intramolecular charge complexation is inhibited by sterics or competitive binding. It is demonstrated with molecular mechanics that significant structural changes occur upon loss of 18C6 in model peptides. Examination of the loss of 18C6 adducts from proteins following collisional activation reveals that lower charge states lose the most 18C6. The degree of 18C6 adduct stability may reflect the degree of structural reorganization that occurs following collisional activation, suggesting that lower charge states represent structures that are not similar to gas phase idealized states. In this regard, 18C6 may serve the function of protecting solution phase protein structure. Collisional activation of holomyoglobin with 18C6 adducts attached reveals that heme loss occurs primarily after 18C6 loss, further supporting the notion that 18C6 protects native structure by solvating charged sites.

Dipolar DC induced collisional activation of non‐dissociated electron‐transfer products

The application of electron transfer and dipolar direct current induced collisional activation (ET‐DDC) for enhanced sequence coverage of peptide/protein cations is described. A DDC potential is applied across one pair of opposing rods in the high‐pressure collision cell of a hybrid quadrupole/time‐of‐flight tandem mass spectrometer (QqTOF) to induce collisional activation, in conjunction with electron transfer reactions. As a broadband technique, DDC can be employed for the simultaneous collisional activation of all the first‐generation charge‐reduced precursor ions (eg, electron transfer no‐dissociation or ETnoD products) from electron transfer reactions over a relatively broad mass‐to‐charge range. A systematic study of ET‐DDC induced collision activation on peptide/protein cations revealed an increase in the variety (and abundances) of sequence informative fragment ions, mainly c‐ and z‐type fragment ions, relative to products derived directly via electron transfer dissociation (ETD). Compared with ETD, which has low dissociation efficiency for low‐charge‐state precursor ions, ET‐DDC also showed marked improvement, providing a sequence coverage of 80% to 85% for all the charge states of ubiquitin. Overall, this method provides a simple means for the broadband collisional activation of ETnoD ions in the same collision cell in which they are generated for improved structural characterization of polypeptide and protein cations subjected to ETD.     

The solvation of Cu2+ with gas-phase clusters of water and ammonia

A detailed study has been undertaken of the gas-phase chemistry of [Cu(H2O)N]2+ and [Cu(NH3)N]2+ complexes. Ion intensity distributions and fragmentation pathways (unimolecular and collision-induced) have been recorded for both complexes out as far as N=20. Unimolecular fragmentation is dominated by Coulomb explosion (separation into two single charged units) on the part of the smaller ions, but switches to neutral molecule loss for N>7. In contrast, collisional activation promotes extensive electron capture from the collision gas, with the appearance of particular singly charged fragment ions being sensitive to the size and composition of the precursor. The results show clear evidence of the unit [Cu(X)8]2+ being of special significance, and it is proposed that the hydrogen-bonded structure associated with this ion is responsible for stabilizing the dipositive charge on Cu2+ in aqueous solution.     

Decompositions of odd- and even-electron anions derived from deoxy-polyadenylates

Radical anions have been formed via electron transfer from multiply charged 5′-d(AAA)-3′ and 5′-d(AAAA)-3′ anions to CCl₃ ⁺. These ions have been isolated in a quadrupole ion trap operated with helium bath gas at a pressure of 1 mtorr and subjected to resonance excitation (i. e., conventional ion trap collisional activation). Collisional activation of the even-electron species of the same charge state formed directly via electrospray was also performed by using essentially identical conditions. The collisional activation data can be compared directly without ambiguity arising from differences in parent ion internal energies and/or dissociation time frames. Both the odd- and even-electron anions yield extensive sequence-informative fragmentation but show significant differences in the extent of nucleobase loss and in the relative contributions from the various sequence diagnostic dissociation channels. The results of this study indicate that radical anions derived from multiply deprotonated oligo-deoxynucleotides that survive the electron transfer process are stable with respect to fragmentation in the ion trap environment under normal storage conditions and that the unimolecular dissociation behavior of these ions differs from the even-electron anions of the same charge state. These findings suggest, therefore, that odd- and even-electron anions might be used to provide complementary sequence information in cases in which neither ion type provides the full sequence.     

Ion trap collision-induced dissociation of locked nucleic acids

Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3′C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics.     

Dissociation of different conformations of ubiquitin ions

The fragmentation pathways of different conformations of three charge states of ubiquitin ions are examined using ion mobility/collisional activation/time-of-flight techniques. Mass spectra for fragments for different conformers of a single charge state appear to be identical (within the experimental reproducibility). These results are consistent with a mechanism in which different conformers of each charge state rearrange to similar dissociation transition states prior to fragment formation.     

Proton transfer reactions for improved peptide characterisation

The combination of deprotonation (via ion/molecule and ion/ion reactions) and low-energy collision-induced dissociation (CID) has been explored for the enhanced characterisation of tryptic peptides via access to different precursor charge states. This approach allows instant access to fragmentation properties of singly and doubly protonated precursors (arising from the availability of mobile protons) in a single experiment. Considering both charge states extended our base of structurally informative data (in comparison with considering just a single charge state) due to generation of additional sequence ions and by obtaining supplementary structural information derived from selective cleavages. Roughly 37% of combined data sets (CID spectra of doubly and singly charged precursor) showed a greater database identification confidence than each set alone. Moreover, comparison between a number of sequence ions of the singly charged precursor and the doubly charged precursor provided a mean of distinguishing the two classes of tryptic peptides (arginine or lysine containing).     

Electron transfer dissociation with supplemental activation to differentiate aspartic and isoaspartic residues in doubly charged peptide cations

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z ^• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.     

High-energy collision-induced dissociation of multiply charged polypeptides produced by electrospray

The recent commercial implementation of an electrospray source on a four-sector mass spectrometer has allowed the study of high-energy collisional activation of multiply charged cations. With this configuration, higher mass-to-charge ratios can be accommodated in both precursor ion selection and fragment ion detection. Good mass accuracy facilitates analysis of fragment ions and allows more reliable mechanistic correlation of these fragments. A convenient scheme was devised to permit the use of kilovolt potentials in both MS-I and MS-II, with precursors of varying charge states. Algorithms were devised to assign masses of different types of multiply charged fragment ions. Nine polypeptides were studied in the mass range 2000–5000 Da. Through this entire mass range, fragment ions were observed to be amply formed by cleavages in both the backbone and side chains, analogous to high-energy collisional activation of singly charged ions. This stands in sharp contrast to the patterns reported with low-energy, multiple collisions. Abundances of sequence ion series are influenced by the positions of basic residues. Analysis of charge distributions in fragment ions also indicates that the charges tend to be spread out across the peptides.     

Ion trap collision-induced dissociation of multiply deprotonated RNA: c/y-ions versus (a-B)/w-ions

The dissociation of model RNA anions has been studied as a function of anion charge state and excitation amplitude using ion trap collisional activation. Similar to DNA anions, the precursor ion charge state of an RNA anion plays an important role in directing the preferred dissociation channels. Generally, the complementary c/y-ions from 5′ P-O bond cleavage dominate at low to intermediate charge states, while other backbone cleavages appear to a limited extent but increase in number and relative abundance at higher excitation energies. The competition between base loss, either as a neutral or as an anion, as well as the preference for the identity of the lost base are also observed to be charge-state dependent. To gain further insight into the partitioning of the dissociation products among the various possible channels, model dinucleotide anions have been subjected to a systematic study. In comparison to DNA, the 2′-OH group on RNA significantly facilitates the dissociation of the 5′ P-O bond. However, the degree of excitation required for a 5′ base loss and the subsequent 3′ C-O bond cleavage are similar for the analogous RNA and DNA dinucleotides. Data collected for protonated dinucleotides, however, suggest that the 2′-OH group in RNA can stabilize the glycosidic bond of a protonated base. Therefore, base loss from low charge state oligonucleotide anions, in which protonation of one or more bases via intramolecular proton transfer can occur, may also be stabilized in RNA anions relative to corresponding DNA anions.     

Charge Transfer and Metastable Ion Dissociation of Multiply Charged Molecular Cations Observed by Using Reflectron Time-of-Flight Mass Spectrometry

A multiply charged molecule expands the range of a mass window and is utilized as a precursor to provide rich sequence coverage; however, reflectron time-of-flight mass spectrometer has not been well applied to the product ion analysis of multiply charged precursor ions. Here, we demonstrate that the range of the mass-to-charge ratio of measurable product ions is limited in the cases of multiply charged precursor ions. We choose C₆F₆ as a model molecule to investigate the reactions of multiply charged molecular cations formed in intense femtosecond laser fields. Measurements of the time-of-flight spectrum of C₆F₆ by changing the potential applied to the reflectron, combined with simulation of the ion trajectory, can identify the species detected behind the reflectron as the neutral species and/or ions formed by the collisional charge transfer. Moreover, the metastable ion dissociations of doubly and triply charged C₆F₆ are identified. The detection of product ions in this manner can diminish interference by the precursor ion. Moreover, it does not need precursor ion separation before product ion analysis. These advantages would expand the capability of mass spectrometry to obtain information about metastable ion dissociation of multiply charged species.     

Dissociation of heme-globin complexes by blackbody infrared radiative dissociation: Molecular specificity in the gas phase?

The temperature dependence of the unimolecular kinetics for dissociation of the heme group from holo-myoglobin (Mb) and holo-hemoglobin alpha-chain (Hb-alpha) was investigated with blackbody infrared radiative dissociation (BIRD). The rate constant for dissociation of the 9 + charge state of Mb formed by electrospray ionization from a "pseudo-native" solution is 60% lower than that of Hb-alpha at each of the temperatures investigated. In solutions of pH 5.5-8.0, the thermal dissociation rate for Mb is also lower than that of HB-alpha (Hargrove, M. S. et al. J. Biol. Chem.1994, 269, 4207-4214). Thus, Mb is thermally more stable with respect to heme loss than Hb-alpha both in the gas phase and in solution. The Arrhenius activation parameters for both dissociation processes are indistinguishable within the current experimental error (activation energy 0.9 eV and pre-exponential factor of 10(8-10) s(-1)). The 9+ to 12+ charge states of Mb have similar Arrhenius parameters when these ions are formed from pseudo-native solutions. In contrast, the activation energies and pre-exponential factors decrease from 0.8 to 0.3 eV and 10(7) to 10(2) s(-1), respectively, for the 9 + to 12 + charge states formed from acidified solutions in which at least 50% of the secondary structure is lost. These results demonstrate that gas-phase Mb ions retain clear memory of the composition of the solution from which they are formed and that these differences can be probed by BIRD.     

Gas-Phase Peptide Sulfinyl Radical Ions: Formation and Unimolecular Dissociation

A variety of peptide sulfinyl radical (RSO?) ions with a well-defined radical site at the cysteine side chain were formed at atmospheric pressure (AP), sampled into a mass spectrometer, and investigated via collision-induced dissociation (CID). The radical ion formation was based on AP reactions between oxidative radicals and peptide ions containing single inter-chain disulfide bond or free thiol group generated from nanoelectrospray ionization (nanoESI). The radical induced reactions allowed large flexibility in forming peptide radical ions independent of ion polarity (protonated or deprotonated) or charge state (singly or multiply charged). More than 20 peptide sulfinyl radical ions in either positive or negative ion mode were subjected to low energy collisional activation on a triple-quadrupole/linear ion trap mass spectrometer. The competition between radical- and charge-directed fragmentation pathways was largely affected by the presence of mobile protons. For peptide sulfinyl radical ions with reduced proton mobility (i.e., singly protonated, containing basic amino acid residues), loss of 62?Da (CH₂SO), a radical-initiated dissociation channel, was dominant. For systems with mobile protons, this channel was suppressed, while charge-directed amide bond cleavages were preferred. The polarity of charge was found to significantly alter the radical-initiated dissociation channels, which might be related to the difference in stability of the product ions in different ion charge polarities.