Electron Capture Dissociation Mass Spectrometry Research Articles

Top-Down Structural Characterization of Native Ubiquitin Combining Solution-Stable Isotope Labeling, Trapped Ion Mobility Spectrometry, and Tandem Electron Capture Dissociation Mass Spectrometry.

Solution-phase hydrogen/deuterium exchange (HDX) coupled to native ion mobility spectrometry mass spectrometry (IMS-MS) can provide complementary structural information about the conformational dynamics of biological molecules. In the present work, the solution-stable isotope labeling (SIL) combined with trapped ion mobility spectrometry (TIMS) in tandem with top-down electron capture dissociation (ECD) is illustrated for the structural characterization of the solution native states of ubiquitin. Four different ubiquitin electrospray solution conditions: (i) single-tip nondeuterated, (ii) theta tip for online SIL HDX, (iii) single-tip SIL-deuterated, and (iv) theta tip for online SIL H/D back exchange (HDbX), were investigated to assess the H/D exchange reactivities of native ubiquitin. The combination of TIMS and ECD in a q-ToF MS instrument allowed for additional inspection of gas-phase HDbX added by top-down fragmentation, revealing the exposed and protected residues with limited scrambling effects (e.g., intramolecular H/D migration). A native charge state distribution (5+ to 7+) and TIMS profiles were observed under the single-tip nondeuterated solution conditions. Mass shift distributions of ∼40, ∼104, and ∼87D were observed when incorporating deuterium for online SIL HDX, SIL HDX, and online SIL HDbX, respectively, while retaining similar conformational states. ECD fragmentation allowed for the localization of the deuterated labeled residues of the peptide fragments, with a sequence coverage of ∼90%, for each of the ubiquitin solution condition. Changes in the TIMS trapping time settings (∼70 to ∼795 ms) were used to determine the H/D back exchange dynamics of native ubiquitin. HDbX-TIMS-q-ECD-MS/MS exhibited H/D back exchanges in the six-residue C-terminal tail as well as around Lys6, Lys11, Lys33, Lys48, and Lys63 residues, indicating that these regions are the most exposed area (less protected hydrogens) of ubiquitin as compared to the rest of the core residues that adopt a compact β-grasp fold (protected hydrogens), which was consistent with the accessible surface area of ubiquitin. The present data highlight for the first time consistency between the solution HDX and gas-phase HDbX-TIMS data for native studies.

Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry.

We report the first sequencing of morpholino antisense oligonucleotides (phosphorodiamidate morpholino oligomers, PMOs) using electron capture dissociation (ECD) mass spectrometry. In this research, we found dissociation of the backbone of 18- to 25-mer PMOs to produce d and z ions as the major ions, and 100% cleavage coverage (sequence coverage) was obtained with these ions. This is a critical contrast with beam-type collision-induced dissociation, which dominantly induces base loss, so it is difficult to obtain sequence information. The results showed that an electron beam energy (typically 15 eV) can be used universally for PMOs with different sequences, lengths, and charge states so that no detailed optimization is required for multiprecursor targeting liquid chromatography coupled with tandem mass spectrometry measurements. We also confirmed that the ECD reaction speed was compatible with the high-performance liquid chromatography time scale. Finally, we demonstrated a liquid chromatography electron capture dissociation tandem mass spectrometry workflow to survey the modification sites of the emulated PMO impurities.

Online protein unfolding characterized by ion mobility electron capture dissociation mass spectrometry: cytochrome C from neutral and acidic solutions.

Electrospray ionization mass spectrometry (ESI-MS) experiments, including ion mobility spectrometry mass spectrometry (ESI-IMS-MS) and electron capture dissociation (ECD) of proteins ionized from aqueous solutions, have been used for the study of solution-like structures of intact proteins. By mixing aqueous proteins with denaturants online before ESI, the amount of protein unfolding can be precisely controlled and rapidly analyzed, permitting the characterization of protein folding intermediates in protein folding pathways. Herein, we mixed various pH solutions online with aqueous cytochrome C for unfolding and characterizing its unfolding intermediates with ESI-MS charge state distribution measurements, IMS, and ECD. The presence of folding intermediates and unfolded cytochrome c structures were detected from changes in charge states, arrival time distributions (ATDs), and ECD. We also compared structures from nondenaturing and denaturing solution mixtures measured under "gentle" (i.e., low energy) ion transmission conditions with structures measured under "harsh" (i.e., higher energy) transmission. This work confirms that when using "gentle" instrument conditions, the gas-phase cytochrome c ions reflect attributes of the various solution-phase structures. However, "harsh" conditions that maximize ion transmission produce extended structures that no longer correlate with changes in solution structure.

Structural Analysis of 14-3-3-ζ-Derived Phosphopeptides Using Electron Capture Dissociation Mass Spectrometry, Traveling Wave Ion Mobility Spectrometry, and Molecular Modeling.

Previously, we have demonstrated the effect of salt bridges on the electron capture dissociation mass spectrometry behavior of synthetic model phosphopeptides and applied an ion mobility spectrometry/molecular modeling approach to rationalize the findings in terms of peptide ion structure. Here, we develop and apply the approach to a biologically derived phosphopeptide. Specifically, we have investigated variants of a 15-mer phosphopeptide VVGARRSsWRVVSSI (s denotes phosphorylated Ser) derived from Akt1 substrate 14-3-3-ζ, which contains the phosphorylation motif RRSsWR. Variants were generated by successive arginine-to-leucine substitutions within the phosphorylation motif. ECD fragmentation patterns for the eight phosphopeptide variants show greater sequence coverage with successive R → L substitutions. Peptides with two or more basic residues had regions with no sequence coverage, while full sequence coverage was observed for peptides with one or no basic residues. For three of the peptide variants, low-abundance fragments were observed between the phosphoserine and a basic residue, possibly due to the presence of multiple conformers with and without noncovalent interactions between these residues. For the five variants whose dissociation behavior suggested the presence of intramolecular noncovalent interactions, we employed ion mobility spectrometry and molecular modeling to probe the nature of these interactions. Our workflow allowed us to propose candidate structures whose noncovalent interactions were consistent with the ECD data for all of the peptides modeled. Additionally, the AMBER parameter sets created for and validated by this work are presented and made available online ( http://www.biosciences-labs.bham.ac.uk/cooper/datasets.php ).

Difference of Electron Capture and Transfer Dissociation Mass Spectrometry on Ni(2+)-, Cu(2+)-, and Zn(2+)-Polyhistidine Complexes in the Absence of Remote Protons.

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) in metal-peptide complexes are dependent on the metal cation in the complex. The divalent transition metals Ni(2+), Cu(2+), and Zn(2+) were used as charge carriers to produce metal-polyhistidine complexes in the absence of remote protons, since these metal cations strongly bind to neutral histidine residues in peptides. In the case of the ECD and ETD of Cu(2+)-polyhistidine complexes, the metal cation in the complex was reduced and the recombination energy was redistributed throughout the peptide to lead a zwitterionic peptide form having a protonated histidine residue and a deprotonated amide nitrogen. The zwitterion then underwent peptide bond cleavage, producing a and b fragment ions. In contrast, ECD and ETD induced different fragmentation processes in Zn(2+)-polyhistidine complexes. Although the N-Cα bond in the Zn(2+)-polyhistidine complex was cleaved by ETD, ECD of Zn(2+)-polyhistidine induced peptide bond cleavage accompanied with hydrogen atom release. The different fragmentation modes by ECD and ETD originated from the different electronic states of the charge-reduced complexes resulting from these processes. The details of the fragmentation processes were investigated by density functional theory. Graphical Abstract ᅟ.

Letter: Evaluation and comparison of collision-induced dissociation and electron-capture dissociation for top-down analysis of intact ribonuclease B.

It has been previously reported that the glycosylation site and protein-sequence information could be obtained for ribonuclease B by top-down electron-capture dissociation (ECD) and collision-induced dissociation (CID) mass spectrometry (MS). However, the sequence coverage of ribonuclease B was limited in a single activation, and the structural information on the glycan moiety was not probed successfully in previous experiments. Here, we demonstrate that ECD and CID techniques can be used together as an effective top- down method for the structural characterization of intact glycoprotein. Even without an elaborate pre- or post- ECD activation, a high sequence coverage (<90%) of ribonuclease B could be achieved with substantial amounts of structural information for the glycan moiety. By comparing our work with previous results, it is postulated that the disulfide bond reduction strategy might play a significant role in determining the efficiency of top-down MS.

Electron capture dissociation mass spectrometry of phosphopeptides: Arginine and phosphoserine

We have previously shown that the presence of phosphorylation can inhibit detection of electron capture dissociation (ECD) fragments of doubly charged peptide ions. The presence of non-covalent interactions, in the form of salt-bridges or ionic hydrogen bonds, prevents the separation of fragments following backbone cleavage. Here, we show the electron capture dissociation mass spectrometry of a suite of model peptides designed to investigate the relationship between phosphoserine and arginine position, namely AApSAnRAmKA (n=0–6, m=6–0), the presence of lysine residues (AApSAAKAARAKA) and AAApSARAAAAKAAAK, and the presence of proline A(A/P)ApSARAAA(A/P)KAAAK. The latter are analogous to the peptides studied previously. The results show that the presence of phosphoserine and basic amino acid residues alone does not inhibit ECD fragmentation, even when the number of basic amino acid residues is greater than the precursor charge state. Neither did the presence of proline in the peptide sequence suppress ECD backbone cleavage. Nevertheless, the presence and relative position of the phosphorylated residue do alter the observed backbone fragmentation abundance. In addition, the presence of phosphorylation appears to inhibit cleavage within the arginine side-chain regardless of the relative position of the arginine residue. The results suggest that ECD fragmentation behaviour is dependent on the three-dimensional structure of a peptide rather than its sequence.

Probing the electron capture dissociation mass spectrometry of phosphopeptides with traveling wave ion mobility spectrometry and molecular dynamics simulations.

Electron capture dissociation mass spectrometry offers several advantages for the analysis of peptides, most notably that backbone c and z fragments typically retain labile modifications such as phosphorylation. We have shown previously that, in some cases, the presence of phosphorylation has a deleterious effect on peptide sequence coverage, and hypothesized that intramolecular interactions involving the phosphate group were preventing separation of backbone fragments. In the present work, we seek to rationalize the observed ECD behavior through a combination of ECD of model peptides, traveling wave ion mobility mass spectrometry and molecular dynamics simulations. The results suggest that for doubly protonated ions of phosphopeptide APLpSFRGSLPKSYVK a salt-bridge structure is favored, whereas for the doubly-protonated ions of APLSFRGSLPKpSYVK ionic hydrogen bonds predominate. Graphical ᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-015-1094-1) contains supplementary material, which is available to authorized users.

Electron capture dissociation of extremely supercharged protein ions formed by electrospray ionisation

Extreme supercharging of proteins yields significant performance gains for the direct characterization of protein sequences by electron capture dissociation mass spectrometry.

On the Viability of Heterolytic Peptide N–Cα Bond Cleavage in Electron Capture and Transfer Dissociation Mass Spectrometry

While frequently employed as an experimental technique, the mechanistic picture surrounding the gas-phase dissociation of peptides carrying multiple positive charges during electron capture and electron transfer dissociation tandem mass spectrometry remains incomplete. Despite this mechanistic uncertainty, most proposals agree that the peptide backbone N-Cα bond located to the C-terminal (right) side of an aminoketyl radical formed in a peptide backbone during the electron capture process is homolytically cleaved. Recently, we introduced the "enol" mechanism, which proposes that a backbone N-Cα bond located to the N-terminal (left) side of an aminoketyl radical is cleaved heterolytically. Here, we further validate this mechanism using replica-exchange molecular dynamics to create unbiased representative sets of low-energy conformers for several model tryptic peptide systems (H-Alax-Lys-OH(2+), x = 3-5). Transition state barrier enthalpies for the cleavage of N-Cα bonds proceeding via the homolytic (right-side) and heterolytic (left-side) pathways, determined by density functional computations, identify the preferred cleavage route for each conformer. These findings support our original hypothesis that heterolytic N-Cα cleavage can exist in a competitive balance with homolytic cleavages, independent of the relative energy of the precursor dication species. Smaller peptide systems see decreased heterolytic N-Cα cleavage probabilities, likely resulting from an insufficient hydrogen-bonding network needed to stabilize and ultimately annihilate the transition state zwitterion. This observation may explain the early dismissal of left-side cleavage pathways based on computational studies employing small model systems.

Gas-Phase Structure of Amyloid-β (12 – 28) Peptide Investigated by Infrared Spectroscopy, Electron Capture Dissociation and Ion Mobility Mass Spectrometry

The gas-phase structures of doubly and triply protonated Amyloid-β12-28 peptides have been investigated through the combination of ion mobility (IM), electron capture dissociation (ECD) mass spectrometry, and infrared multi-photon dissociation (IRMPD) spectroscopy together with theoretical modeling. Replica-exchange molecular dynamics simulations were conducted to explore the conformational space of these protonated peptides, from which several classes of structures were found. Among the low-lying conformers, those with predicted diffusion cross-sections consistent with the ion mobility experiment were further selected and their IR spectra simulated using a hybrid quantum mechanical/semiempirical method at the ONIOM DFT/B3LYP/6-31g(d)/AM1 level. In ECD mass spectrometry, the c/z product ion abundance (PIA) has been analyzed for the two charge states and revealed drastic differences. For the doubly protonated species, N - Cα bond cleavage occurs only on the N and C terminal parts, while a periodic distribution of PIA is clearly observed for the triply charged peptides. These PIA distributions have been rationalized by comparison with the inverse of the distances from the protonated sites to the carbonyl oxygens for the conformations suggested from IR and IM experiments. Structural assignment for the amyloid peptide is then made possible by the combination of these three experimental techniques that provide complementary information on the possible secondary structure adopted by peptides. Although globular conformations are favored for the doubly protonated peptide, incrementing the charge state leads to a conformational transition towards extended structures with 310- and α-helix motifs.

ChemInform Abstract: Principles of Electron Capture and Transfer Dissociation Mass Spectrometry Applied to Peptide and Protein Structure Analysis

AbstractReview: 60 refs.

Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis

This tutorial review describes the principles and practices of electron capture and transfer dissociation (ECD/ETD or ExD) mass spectrometry (MS) employed for peptide and protein structure analysis. ExD MS relies on interactions between gas phase peptide or protein ions carrying multiple positive charges with either free low-energy (~1 eV) electrons (ECD), or with reagent radical anions possessing an electron available for transfer (ETD). As a result of recent implementation on sensitive, high resolution, high mass accuracy, and liquid chromatography timescale-compatible mass spectrometers, ExD, more specifically, ETD MS has received particular interest in life science research. In addition to describing the fundamental aspects of ExD radical ion chemistry, this tutorial provides practical guidelines for peptide de novo sequencing with ExD MS, as well as reviews some of the current capabilities and limitations of these techniques. The merits of ExD MS are discussed primarily within the context of life science research.

Spatial extent of fragment-ion abundances in electron transfer dissociation and electron capture dissociation mass spectrometry of peptides

Earlier work from the first author's group has suggested that, in electron capture dissociation (ECD) or electron transfer dissociation (ETD) mass spectrometry experiments, an electron is initially attached into a Rydberg orbital centered at one of the peptide's positive sites (likely a protonated N-terminus, Lysine, Arginine, or Histidine). Moreover, this earlier work predicted that only Rydberg orbitals having principal quantum numbers n=3–6 are populated in ECD and only n=3 and 4 in ETD (when an anion donor having an electron binding energy of ca. 0. 6eV is used), and that the populations of these levels are very similar in the nascent charge-reduced peptide. Based upon these predictions, the present paper develops a framework for predicting the abundances of closed-shell c and open-shell z fragments as a function of distance along the backbone from the site initially holding the attached electron in a Rydberg orbital. The framework is not aimed at differences in branching ratios caused by differences in the physical properties of side chains along the backbone but on the spatial distances between the charged site holding the electron and the backbone amide units. The predictions of this model are tested using ECD and ETD data from derived from experiments carried out using simultaneous infrared photo-activation of the parent ions. Such activated-ion (AI) experiments are thought to disrupt much of the parent ion's secondary structure, which we believe allows us to make more reliable estimates of distances between the charged sites and the various amino acids’ amide groups. The abundance patterns predicted based upon the framework described herein are found to be reasonably consistent with the experimental data. However, the data also provide evidence that internal solvation of the peptide's charged sites remains intact even under AI conditions, and that some of these solvated-ion structures (those involving a charged Lys or N-terminal amine) contribute incrementally to the abundances of fragment ions arising from cleaving nearby NCα bonds. As a result, we conclude that ECD and ETD fragment ion abundances are determined by a combination of factors: (i) internal solvation of charged sites, (ii) spatial distributions or Rydberg orbitals charge densities within several residues of charged sites, and (iii) variations induced by differences in physical properties of side chains. It is primarily the first two of these three that the present paper addresses.

Liquid Chromatography–Atmospheric Pressure Electron Capture Dissociation Mass Spectrometry for the Structural Analysis of Peptides and Proteins

Atmospheric pressure electron capture dissociation (AP-ECD) is an emerging technique with the potential to be a more accessible alternative to conventional ECD/electron transfer dissociation (ETD) methods because it can be implemented using a stand-alone ion source device suitable for use with any existing or future electrospray ionization mass spectrometer. With AP-ECD, no modification of the main instrument is required, so it may easily be retrofitted to instruments not originally equipped with ECD/ETD capabilities. Here, we present our first purpose-built AP-ECD source and demonstrate its use in conjunction with capillary LC for the analysis of substance P, a tryptic digest of bovine serum albumin, and a phosphopeptide mixture. Quality ECD spectra were obtained for all the samples at the low femtomole level, proving that LC-AP-ECD-MS is suitable for the structural analysis of peptides and protein digests, in this case using an unmodified quadrupole time-of-flight mass spectrometer built ca. 2002.

Structural Interrogation of Electrosprayed Peptide Ions by Gas-Phase H/D Exchange and Electron Capture Dissociation Mass Spectrometry

The structural characterization of gaseous biomolecular ions remains a challenging task. Here, we employ a combination of gas-phase hydrogen-deuterium exchange (HDX) and electron capture dissociation (ECD) mass spectrometry for gaining insights into the properties of two electrosprayed peptides: RA(9)K and RG(9)K. Mass analysis of ECD fragments provides spatially resolved labeling information. ND(3)-mediated HDX at peptide termini and amino acid side chains goes to completion within 1 s. Backbone amide labeling occurs more slowly, and proceeds in a structurally sensitive fashion. HDX is more extensive for RG(9)K than for RA(9)K, suggesting a more "open" conformation for the former. Residues 7-10 in RA(9)K are strongly protected, which indicates the presence of stable backbone hydrogen bonds at these sites. Our findings are consistent with the results of previous ion mobility measurements and computational investigations. Overall, it appears that the combination of gas-phase HDX and ECD represents a viable approach for uncovering structural features of biomolecular ions in the gas phase.

Repeatability and Reproducibility of Product Ion Abundances in Electron Capture Dissociation Mass Spectrometry of Peptides

Site-specific reproducibility and repeatability of electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) are of fundamental importance for product ion abundance (PIA)-based peptide and protein structure analysis. However, despite the growing interest in ECD PIA-based applications, these parameters have not yet been investigated in a consistent manner. Here, we first provide a detailed description of the experimental parameters for ECD-based tandem mass spectrometry performed on a hybrid linear ion trap (LTQ) FT-ICR MS. In the following, we describe the evaluation and comparison of ECD and infrared multiphoton dissociation (IRMPD) PIA methodologies upon variation of a number of experimental parameters, for example, cathode potential (electron energy), laser power, electron and photon irradiation periods and pre- irradiation delays, as well as precursor ion number. Ranges of experimental parameters that yielded an average PIA variation below 5% and 15% were determined for ECD and IRMPD, respectively. We report cleavage site-dependent ECD PIA variation below 20% and correlation coefficients between fragmentation patterns superior to 0.95 for experiments performed on three FT-ICR MS instruments. Overall, the encouraging results obtained for ECD PIA reproducibility and repeatability support the use of ECD PIA as a complementary source of information to m/z data in radical-induced dissociation applied for peptide and protein structure analysis.

Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies

The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrometry and its coupling with native electrospray ionization (ESI) make the combination complementary to other biophysical methods of protein analysis. Protein assemblies with molecular masses up to MDa are now accessible by this approach. Most current approaches have used quadrupole/time-of-flight tandem mass spectrometry, sometimes coupled with ion mobility, to reveal stoichiometry, shape, and dissociation of protein assemblies. The amino-acid sequence of the subunits, however, still relies heavily on independent bottom-up proteomics. We describe here an approach to study protein assemblies that integrates electron-capture dissociation (ECD), native ESI, and FTICR mass spectrometry (12 T). Flexible regions of assembly subunits of yeast alcohol dehydrogenase (147 kDa), concanavalin A (103 kDa), and photosynthetic Fenna-Matthews-Olson antenna protein complex (140 kDa) can be sequenced by ECD or "activated-ion" ECD. Furthermore, noncovalent metal-binding sites can also be determined for the concanavalin A assembly. Most importantly, the regions that undergo fragmentation, either from one of the termini by ECD or from the middle of a protein, as initiated by CID, correlate well with the B-factor from X-ray crystallography of that protein. This factor is a measure of the extent an atom can move from its coordinated position as a function of temperature or crystal imperfections. The approach provides not only top-down proteomics information of the complex subunits but also structural insights complementary to those obtained by ion mobility.

Conformer-Specific Hydrogen Exchange Analysis of Aβ(1–42) Oligomers by Top-Down Electron Capture Dissociation Mass Spectrometry

Protein structural studies are particularly challenging under conditions in which several conformational species (e.g., monomers and aggregated forms) coexist in solution. Most spectroscopic techniques provide population-averaged data. Hence, it is usually not possible to obtain detailed structural information on individual protein species in heterogeneous samples. The current work employs an experimental strategy that addresses this issue. Solution-phase hydrogen exchange (HX) is used in combination with tandem mass spectrometry. Electrosprayed intact ions exhibiting specific HX mass shifts are selected in the gas phase, followed by electron capture dissociation. The resulting fragment ion deuteration pattern provides amide hydrogen bonding information in a conformer-specific and spatially resolved fashion. The feasibility of this approach is demonstrated by applying it to neurotoxic Aβ(1-42) oligomers that coexist with disordered monomers in solution. The findings of this study point to similarities between oligomers and mature amyloid fibrils with regard to the Aβ(1-42) backbone organization. Specifically, fibrils and oligomers appear to share a β-loop-β secondary structure motif. The spatial resolution obtained with the "top-down" approach used here exceeds that of earlier proteolysis-based HX data on Aβ.

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I.

5'-AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is activated when cellular AMP to ATP ratios rise, potentially serving as a key regulator of cellular energetics. Among the known targets of AMPK are catabolic and anabolic enzymes, but little is known about the ability of this kinase to phosphorylate myofilament proteins and thereby regulating the contractile apparatus of striated muscles. Here, we demonstrate that troponin I isoforms of cardiac (cTnI) and fast skeletal (fsTnI) muscles are readily phosphorylated by AMPK. For cTnI, two highly conserved serine residues were identified as AMPK sites using a combination of high-resolution top-down electron capture dissociation mass spectrometry, (32) P-incorporation, synthetic peptides, phospho-specific antibodies, and site-directed mutagenesis. These AMPK sites in cTnI were Ser149 adjacent to the inhibitory loop and Ser22 in the cardiac-specific N-terminal extension, at the level of cTnI peptides, the intact cTnI subunit, whole cardiac troponin complexes and skinned cardiomyocytes. Phosphorylation time-course experiments revealed that Ser149 was the preferred site, because it was phosphorylated 12-16-fold faster than Ser22 in cTnI. Ser117 in fsTnI, analogous to Ser149 in cTnI, was phosphorylated with similar kinetics as cTnI Ser149. Hence, the master energy-sensing protein AMPK emerges as a possibly important regulator of cardiac and skeletal contractility via phosphorylation of a preferred site adjacent to the inhibitory loop of the thin filament protein TnI.