Electron Capture Dissociation Research Articles

New Insights into the Intrinsic Electron-Based Dissociation Behavior of Cytochrome c Oligomers.

Between 2003 and 2017, four reports were published that demonstrated the intrinsic ability of the native iron-containing proteins cytochrome c and ferritin to undergo radical-based backbone fragmentation in the gas phase without the introduction of exogenous electrons. For cytochrome c in particular, this effect has so far only been reported to occur in the ion source, preventing the in-depth study of reactions occurring after gas-phase isolation of specific precursors. Here, we report the first observation of this intrinsic native electron capture dissociation behavior after quadrupole isolation of specific charge states of the cytochrome c dimer and trimer, providing direct experimental support for key aspects of the mechanism proposed 20 years ago. Furthermore, we provide evidence that, in contrast to some earlier proposals, these oligomeric states are formed in bulk solution rather than during the electrospray ionization process and that the observed fragmentation site preferences can be rationalized through the structure and interactions within these native oligomers rather than the monomer. We also show that the observed fragmentation pattern─and indeed, whether or not fragmentation occurs─is highly sensitive to the provenance and history of the protein samples, to the extent that samples can show distinct fragmentation behavior despite behaving identically in ion mobility experiments. This rather underexplored method therefore represents an exquisitely sensitive conformational probe and will hopefully receive more attention from the biomolecular mass spectrometry community in the future.

Analysis of Sialic Acid Linkage in N-Linked Glycopeptides Using Liquid Chromatography-Electron-Activated Dissociation Time-of-Flight Mass Spectrometry.

Herein, we report a novel liquid chromatography coupled with tandem mass spectrometry method to characterize N-acetylneuraminic acid (Neu5Ac, Sa) linkage in N-linked glycans in glycopeptides with no sialic acid derivatization. First, we established a separation in reversed-phase high-performance liquid chromatography (HPLC) using a higher formic acid concentration in the mobile phases, which separated the N-glycopeptides depending on the Sa linkage. We also demonstrated a novel characterization method of Sa linkages in N-glycopeptides using electron-activated dissociation. We found that hot electron capture dissociation using an electron beam energy higher than 5 eV cleaved glycosidic bonds in glycopeptides, resulting in each glycosidic bond in the antennas being broken on both sides of the oxygen atom. Such glycosidic bond cleavage at the reducing end (C-type ion) showed the difference in Sa linkages between Sa-Gal, Gal-GlcNAc, and GlcNAc-Man. We proposed a rule to characterize the Sa linkages using the Sa-Gal products. This method was applied to N-glycopeptides in tryptic fetuin digest separated by an optimized reversed-phase HPLC. We successfully identified a number of isomeric glycoforms in the glycopeptides with different Sa links, whose peptide backbones were also simultaneously sequenced by hot ECD.

Determining the Location of the α-Synuclein Dimer Interface Using Native Top-Down Fragmentation and Isotope Depletion-Mass Spectrometry

α-Synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson's disease (PD). Due to its association with PD, αSyn is studied extensively; however, the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn.

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.

De novo glycan sequencing by electronic excitation dissociation MS2-guided MS3 analysis on an Omnitrap-Orbitrap hybrid instrument.

Comprehensive de novo glycan sequencing remains an elusive goal due to the structural diversity and complexity of glycans. Present strategies employing collision-induced dissociation (CID) and higher energy collisional dissociation (HCD)-based multi-stage tandem mass spectrometry (MSn) or MS/MS combined with sequential exoglycosidase digestions are inherently low-throughput and difficult to automate. Compared to CID and HCD, electron transfer dissociation (ETD) and electron capture dissociation (ECD) each generate more cross-ring cleavages informative about linkage positions, but electronic excitation dissociation (EED) exceeds the information content of all other methods and is also applicable to analysis of singly charged precursors. Although EED can provide extensive glycan structural information in a single stage of MS/MS, its performance has largely been limited to FTICR MS, and thus it has not been widely adopted by the glycoscience research community. Here, the effective performance of EED MS/MS was demonstrated on a hybrid Orbitrap-Omnitrap QE-HF instrument, with high sensitivity, fragmentation efficiency, and analysis speed. In addition, a novel EED MS2-guided MS3 approach was developed for detailed glycan structural analysis. Automated topology reconstruction from MS2 and MS3 spectra could be achieved with a modified GlycoDeNovo software. We showed that the topology and linkage configurations of the Man9GlcNAc2 glycan can be accurately determined from first principles based on one EED MS2 and two CID-EED MS3 analyses, without reliance on biological knowledge, a structure database or a spectral library. The presented approach holds great promise for autonomous, comprehensive and de novo glycan sequencing.

Top-"Double-Down" Mass Spectrometry of Histone H4 Proteoforms: Tandem Ultraviolet-Photon and Mobility/Mass-Selected Electron Capture Dissociations.

Post-translational modifications (PTMs) on intact histones play a major role in regulating chromatin dynamics and influence biological processes such as DNA transcription, replication, and repair. The nature and position of each histone PTM is crucial to decipher how this information is translated into biological response. In the present work, the potential of a novel tandem top-"double-down" approach─ultraviolet photodissociation followed by mobility and mass-selected electron capture dissociation and mass spectrometry (UVPD-TIMS-q-ECD-ToF MS/MS)─is illustrated for the characterization of HeLa derived intact histone H4 proteoforms. The comparison between q-ECD-ToF MS/MS spectra and traditional Fourier-transform-ion cyclotron resonance-ECD MS/MS spectra of a H4 standard showed a similar sequence coverage (∼75%) with significant faster data acquisition in the ToF MS/MS platform (∼3 vs ∼15 min). Multiple mass shifts (e.g., 14 and 42 Da) were observed for the HeLa derived H4 proteoforms for which the top-down UVPD and ECD fragmentation analysis were consistent in detecting the presence of acetylated PTMs at the N-terminus and Lys5, Lys8, Lys12, and Lys16 residues, as well as methylated, dimethylated, and trimethylated PTMs at the Lys20 residue with a high sequence coverage (∼90%). The presented top-down results are in good agreement with bottom-up TIMS ToF MS/MS experiments and allowed for additional description of PTMs at the N-terminus. The integration of a 213 nm UV laser in the present platform allowed for UVPD events prior to the ion mobility-mass precursor separation for collision-induced dissociation (CID)/ECD-ToF MS. Selected c305+ UVPD fragments, from different H4 proteoforms (e.g., Ac + Me2, 2Ac + Me2 and 3Ac + Me2), exhibited multiple IMS bands for which similar CID/ECD fragmentation patterns per IMS band pointed toward the presence of conformers, adopting the same PTM distribution, with a clear assignment of the PTM localization for each of the c305+ UVPD fragment H4 proteoforms. These results were consistent with the biological "zip" model, where acetylation proceeds in the Lys16 to Lys5 direction. This novel platform further enhances the structural toolbox with alternative fragmentation mechanisms (UVPD, CID, and ECD) in tandem with fast, high-resolution mobility separations and shows great promise for global proteoform analysis.

Application of Multiple Length Cross-linkers to the Characterization of Gaseous Protein Structure.

The speed, sensitivity, and tolerance of heterogeneity, as well as the kinetic trapping of solution-like states during electrospray, make native mass spectrometry an attractive method to study protein structure. Increases in the resolution of ion mobility measurements and in mass resolving power and range are leading to the increase of the information content of intact protein measurements and an expanded role of mass spectrometry in structural biology. Herein, a suite of different length noncovalent (sulfonate to positively charged side chain) cross-linkers was introduced via gas-phase ion/ion chemistry and used to determine distance restraints of kinetically trapped gas-phase structures of native-like cytochrome c ions. Electron capture dissociation allowed for the identification of cross-linked sites. Different length linkers resulted in distinct pairs of side chains being linked, supporting the ability of gas-phase cross-linking to be structurally specific. The gas-phase lengths of the cross-linkers were determined by conformational searches and density functional theory, allowing for the interpretation of the cross-links as distance restraints. These distance restraints were used to model gas-phase structures with molecular dynamics simulations, revealing a mixture of structures with similar overall shape/size but distinct features, thereby illustrating the kinetic trapping of multiple native-like solution structures in the gas phase.

Localization of Multiple O-Linked Glycans Exhibited in Isomeric Glycopeptides by Hot Electron Capture Dissociation.

We describe a method to obtain a comprehensive profile of multiple glycosylations in glycopeptide isoforms. We detected a wide range of abundances of various O-glycoforms in isomeric glycopeptides using hot electron capture dissociation (hot ECD) in liquid chromatography-tandem mass spectrometry. To capture low abundant glycosylated species, a prototype of a ZenoTOF 7600 system incorporating an efficient electron-activated dissociation device to perform hot ECD was operated in targeted or scheduled high-resolution multiple reaction monitoring workflows. In addition, Zeno trap pulsing was activated to enhance the sensitivity of the time-of-flight mass spectrometer. Sixty-nine O-glycopeptides of the long O-glycopeptides in tryptic bovine fetuin digest were obtained with a relative abundance range from 100 to 0.2%, which included sialylated glycans with Neu5Ac and Neu5Gc.

Exploring Gas-Phase MS Methodologies for Structural Elucidation of Branched N-Glycan Isomers.

Structural isomers of N-glycans that are identical in mass and atomic composition provide a great challenge to conventional mass spectrometry (MS). This study employs additional dimensions of structural elucidation including ion mobility (IM) spectroscopy coupled to hydrogen/deuterium exchange (HDX) and electron capture dissociation (ECD) to characterize three main A2 N-glycans and their conformers. A series of IM-MS experiments were able to separate the low abundance N-glycans and their linkage-based isomers (α1-3 and α1-6 for A2G1). HDX-IM-MS data indicated the presence of multiple gas-phase structures for each N-glycan including the isomers of A2G1. Identification of A2G1 isomers by their collision cross section was complicated due to the preferential collapse of sugars in the gas phase, but it was possible by further ECD fragmentation. The cyclic IM-ECD approach was capable of assigning and identifying each isomer to its IM peak. Two unique cross-ring fragments were identified for each isomer: m/z = 624.21 for α1-6 and m/z = 462.16 for α1-3. Based on these key fragments, the first IM peak, indicating a more compact conformation, was assigned to α1-3 and the second IM peak, a more extended conformer, was assigned to α1-6.

Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation.

Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissociation, electron-transfer dissociation, and electron-capture dissociation combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.

Tandem Mass Spectrometry for Structural Characterization of Doubly-Charged N-Linked Glycopeptides

Three dissociation methods, including collision-induced dissociation (CID), electron capture dissociation (ECD), and electronic excitation dissociation (EED), were systematically compared for structural characterization of doubly charged glycopeptide. CID produced distinctively different tandem mass spectra for glycopeptide adducted with different charge carriers. Protonated species produced mainly glycosidic cleavages in high abundance. CID of magnesiated glycopeptide formed more cross-ring cleavages, whereas doubly sodiated species produced cleavages at both glycan and peptide moieties. The effect of charge carriers on the fragmentation in ECD and EED was lower than that in CID. ECD produced mainly peptide backbone cleavages but limited cleavages at the glycan moiety, whereas EED of glycopeptide resulted in extensive fragmentation throughout the molecular ion regardless of the charge carriers. Magnesiated species gave, however, more cross-ring cleavages than other charge carriers did. These results demonstrated that EED of magnesiated species could be used as a one-step dissociation method for comprehensive structural analysis of glycopeptides.

Multiplexed Conformationally Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation.

In this article, we present an approach for conformationally multiplexed, localized hydrogen deuterium exchange (HDX) of gas-phase protein ions facilitated by ion mobility (IM) followed by electron capture dissociation (ECD). A quadrupole-IM-time of flight instrument previously modified to enable ECD in transmission mode (without ion trapping) immediately following a mobility separation was further modified to allow for deuterated ammonia (ND3) to be leaked in after m/z selection. Collisional activation was minimized to prevent deuterium scrambling from giving structurally irrelevant results. Gas-phase HDX with ECD fragmentation for exchange site localization was demonstrated with the extensively studied protein folding models ubiquitin and cytochrome c. Ubiquitin was ionized from conditions that stabilize the native state and conditions that stabilize the partially folded A-state. IM of deuterated ubiquitin 6+ ions allowed the separation of more compact conformers from more extended conformers. ECD of the separated subpopulations revealed that the more extended (later arriving) conformers had significant, localized differences in the amount of HDX observed. The 5+ charge state showed many regions with protection from HDX, and the 11+ charge state, ionized from conditions that stabilize the A-state, showed high levels of deuterium incorporation throughout most of the protein sequence. The 7+ ions of cytochrome c ionized from aqueous conditions showed greater HDX with unstructured regions of the protein relative to interior, structured regions, especially those involved in heme binding. With careful tuning and attention to deuterium scrambling, our approach holds promise for determining region-specific information on a conformer-selected basis for gas-phase protein structures, including localized characterizations of ligand, epitope, and protein-protein binding.

Multimodal Tandem Mass Spectrometry Techniques for the Analysis of Phosphopeptides.

Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging.

Dissociation of Mannose-Rich Glycans Using Collision-Based and Electron-Based Ion Activation Methods.

Three dissociation methods, including collision-induced dissociation (CID), electron capture dissociation (ECD), and electronic excitation dissociation (EED), were evaluated for the dissociation of doubly charged glycans using sodium or magnesium ions as charge carriers. CID produced mainly glycosidic cleavages, although more cross-ring fragment ions could be obtained at higher intensities when magnesium ions were used as charge carriers [M + Mg]2+. The 0,2A3, 0,3A3, and 0,4A3 ions provided structural information on the 3 → 1 and 6 → 1 linkages of the mannoses. Some internal fragment ions, such as 2,4A5_Y3β, were also produced in high abundance, thus providing additional information on the glycan structure. ECD produced limited fragments compared to other dissociation methods when either of the metal ions were used as charge carriers. Cross-ring fragments were obtained in relatively high abundance, with the charge mainly retained on the nonreducing end. EED produced extensive glycosidic and cross-ring cleavages when either metal charge carrier was used. A higher fragmentation efficiency was achieved and more structural-specific fragments were produced when Na+ was used as the charge carrier. Of the 31 possible cross-ring cleavages, including 0,2-, 0,4-, 1,5-, 2,4-, and 3,5-cleavages, 25 were found, thus providing extensive linkage information. A wide range of fragment ions could be obtained in all dissociation methods when Mg2+ was used as the charge carrier. Two specific analytical approaches were found to produce extensively structural-specific information on the glycans studied, namely CID of magnesiated glycans and EED of sodiated glycans. These two methods were selected to further analyze the larger mannose-rich glycans Man6GlcNAc2 and Man8GlcNAc2 and generated extensive structural information.

Microflow Liquid Chromatography Coupled to Mass Spectrometry (μLC-MS) Workflow for O-Glycopeptides Isomers Analysis Combining Differential Mobility Spectrometry and Collision Induced and Electron Capture Dissociation.

Peptide and protein O-glycosylation can occur mostly on any serine or threonine and could generate several positional isomers, which may coelute during liquid chromatography (LC) separation, challenging their characterization. Ion mobility has emerged as a technique of interest to separate isomeric compounds. In the different ion mobility techniques, differential ion mobility (DMS) includes the particular interest to tune ion separation by the possible addition of an organic modifier. Different microflow liquid chromatography coupled to mass spectrometry (μLC-MS) workflows were investigated for the analysis of a set of four model peptides made of three isomeric glycopeptides and a corresponding nonglycosylated peptide using differential mobility spectrometry (DMS), collision induced dissociation (CID), and electron capture dissociation (ECD). Neither DMS nor LC provided sufficient separation of the three isomeric O-glycopeptides while the nonmodified one was clearly separated by LC. The hyphenation of LC with DMS led to differentiating the three glycopeptides, and further detection and characterization (ECD/CID) with a chimeric collision cell were achieved in a single LC run. The position of the modification was determined from ECD data, while CID data characterized the sugar through its distinctive oxoniums ions in the low mass range.

Spatially Resolved Neuropeptide Characterization from Neuropathological Formalin-Fixed, Paraffin-Embedded Tissue Sections by a Combination of Imaging MALDI FT-ICR Mass Spectrometry Histochemistry and Liquid Extraction Surface Analysis-Trapped Ion Mobility Spectrometry-Tandem Mass Spectrometry.

To make the vast collections of well-documented human clinical samples archived in biobanks accessible for mass spectrometry imaging (MSI), recent developments have focused on the label-free top-down MS analysis of neuropeptides in sections of formalin-fixed, paraffin-embedded (FFPE) tissues. In analogy to immunohistochemistry (IHC), this variant of MSI has been designated MSHC (mass spectrometry histochemistry). Besides the detection and localization of neuropeptide and other biomolecular MS signals in these FFPE samples, there is great interest in their molecular identification and full characterization. We here used matrix assisted laser desorption ionization (MALDI) MSI employing ultrahigh-resolution FT-ICR MS on 2,5-dihydroxybenzoic acid (DHB) coated five-micron sections of human FFPE pituitary to demonstrate clear isotope patterns and elemental composition assignment of neuropeptides (with ∼1 ppm mass accuracy). Besides tandem MS fragmentation pattern analysis to deduce or confirm amino acid sequence information (Arg-vasopressin for the case presented here), there is a need for orthogonal primary structure characterization of the peptide-like MS signals of biomolecules desorbed directly off FFPE tissue sections. In the present work, we performed liquid extraction surface analysis (LESA) extractions on consecutive (uncoated) tissue slices. This enables the successful characterization by ion mobility MS of vasopressin present in FFPE material. Differences in sequence coverage are discussed on the basis of the mobility selected collision induced dissociation (CID), electron capture dissociation (ECD), and UV photodissociation (UVPD) MS/MS. Using Arg-vasopressin as model case (a peptide with a disulfide bridged ring structure), we illustrate the use of LESA in combination with a reduction agent for effective sequencing using mobility selected CID, ECD, and UVPD MS/MS.

Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.

Tandem mass spectrometry of denatured, multiply charged high mass protein precursor ions yield extremely dense spectra with hundreds of broad and overlapping product ion isotopic distributions of differing charge states that yield an elevated baseline of unresolved “noise” centered about the precursor ion. Development of mass analyzers and signal processing methods to increase mass resolving power and manipulation of precursor and product ion charge through solution additives or ion–ion reactions have been thoroughly explored as solutions to spectral congestion. Here, we demonstrate the utility of electron capture dissociation (ECD) coupled with high-resolution cyclic ion mobility spectrometry (cIMS) to greatly increase top-down protein characterization capabilities. Congestion of protein ECD spectra was reduced using cIMS of the ECD product ions and “mobility fractions”, that is, extracted mass spectra for segments of the 2D mobiligram (m/z versus drift time). For small proteins, such as ubiquitin (8.6 kDa), where mass resolving power was not the limiting factor for characterization, pre-IMS ECD and mobility fractions did not significantly increase protein sequence coverage, but an increase in the number of identified product ions was observed. However, a dramatic increase in performance, measured by protein sequence coverage, was observed for larger and more highly charged species, such as the +35 charge state of carbonic anhydrase (29 kDa). Pre-IMS ECD combined with mobility fractions yielded a 135% increase in the number of annotated isotope clusters and a 75% increase in unique product ions compared to processing without using the IMS dimension. These results yielded 89% sequence coverage for carbonic anhydrase.

Determining Copper and Zinc Content in Superoxide Dismutase Using Electron Capture Dissociation Under Native Spray Conditions.

Localizing metal binding to specific sites in proteins remains a challenging analytical problem in vitro and in vivo. Although metal binding can be maintained by "native" electrospray ionization with intact proteins for quantitation by mass spectrometry, subsequent fragmentation of proteins with slow-heating methods like collision-induced dissociation (CID) can scramble and detach metals. In contrast, electron capture dissociation (ECD) fragmentation produces highly localized bond cleavage that is well known to preserve posttranslational modifications. We show how a newly available ECD tool that can be retrofitted on standard QTOF mass spectrometers allows the sites of copper and zinc binding to be localized in the antioxidant enzyme Cu, Zn superoxide dismutase (SOD1). The loss of zinc from Cu, Zn SOD1 has been shown to induce motor neuron death and could have a causal role in the fatal neurodegenerative disease, amyotrophic lateral sclerosis (ALS). The methods described enable copper loss to be distinguished from zinc using distinct ECD fragments of SOD1 and are broadly applicable to other metalloproteins.

Extending Native Top-Down Electron Capture Dissociation to MDa Immunoglobulin Complexes Provides Useful Sequence Tags Covering Their Critical Variable Complementarity-Determining Regions.

Native top-down mass spectrometry (MS) is gaining traction for the analysis and sequencing of intact proteins and protein assemblies, giving access to their mass and composition, as well as sequence information useful for identification. Herein, we extend and apply native top-down MS, using electron capture dissociation, to two submillion Da IgM- and IgG-based oligomeric immunoglobulins. Despite structural similarities, these two systems are quite different. The ∼895 kDa noncovalent IgG hexamer consists of six IgG subunits hexamerizing in solution due to three specifically engineered mutations in the Fc region, whereas the ∼935 kDa IgM oligomer results from the covalent assembly of one joining (J) chain and 5 IgM subunits into an asymmetric “pentamer” stabilized by interchain disulfide bridges. Notwithstanding their size, structural differences, and complexity, we observe that their top-down electron capture dissociation spectra are quite similar and straightforward to interpret, specifically providing informative sequence tags covering the highly variable CDR3s and FR4s of the Ig subunits they contain. Moreover, we show that the electron capture dissociation fragmentation spectra of immunoglobulin oligomers are essentially identical to those obtained for their respective monomers. Demonstrated for recombinantly produced systems, the approach described here opens up new prospects for the characterization and identification of IgMs circulating in plasma, which is important since IgMs play a critical role in the early immune response to pathogens such as viruses and bacteria.

Analysis of Fragmentation Pathways of Peptide Modified with Quaternary Ammonium and Phosphonium Group as Ionization Enhancers.

Peptide modification by a quaternary ammonium group containing a permanent positive charge is a promising method of increasing the ionization efficiency of the analyzed compounds, making ultra-sensitive detection even at the attomolar level possible. Charge-derivatized peptides may undergo both charge remote (ChR) and charge-directed (ChD) fragmentation. A series of model peptide conjugates derivatized with N,N,N-triethyloammonium (TEA), 1-azoniabicyclo[2.2.2]octane (ABCO), 2,4,6-triphenylopyridinium (TPP) and tris(2,4,6-trimetoxyphenylo)phosphonium (TMPP) groups were analyzed by their fragmentation pathways both in collision-induced dissociation (CID) and electron-capture dissociation (ECD) mode. The effect of the fixed-charge tag type and peptide sequence on the fragmentation pathways was investigated. We found that the aspartic acid effect plays a crucial role in the CID fragmentation of TPP and TEA peptide conjugates whereas it was not resolved for the peptides derivatized with the phosphonium group. ECD spectra are mostly dominated by cn ions. ECD fragmentation of TMPP-modified peptides results in the formation of intense fragments derived from this fixed-charge tag, which may serve as reporter ion.