Electron Capture Dissociation Spectra Research Articles

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.

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.

Generating Informative Sequence Tags from Antigen-Binding Regions of Heavily Glycosylated IgA1 Antibodies by Native Top-Down Electron Capture Dissociation.

Immunoglobulins A (IgA) include some of the most abundant human antibodies and play an important role in defending mucosal surfaces against pathogens. The unique structural features of the heavy chain of IgA subclasses (called IgA1 and IgA2) enable them to polymerize via the joining J-chain, resulting in IgA dimers but also higher oligomers. While secretory sIgA oligomers are dominant in milk and saliva, IgAs exist primarily as monomers in serum. No method currently allows disentangling the millions of unique IgAs potentially present in the human antibody repertoire. Obtaining unambiguous sequence reads of their hypervariable antigen-binding regions is a prerequisite for IgA identification. We here report a mass spectrometric method that uses electron capture dissociation (ECD) to produce straightforward-to-read sequence ladders of the variable parts of both the light and heavy chains of IgA1s, in particular, of the functionally critical CDR3 regions. We directly compare the native top-down ECD spectra of a heavily and heterogeneously N- and O-glycosylated anti-CD20 IgA1, the corresponding N-glycosylated anti-CD20 IgG1, and their Fab parts. We show that while featuring very different MS1 spectra, the native top-down ECD MS2 spectra of all four species are nearly identical, with cleavages occurring specifically within the CDR3 and FR4 regions of both the heavy and light chain. From the sequence-informative ECD data of an intact glycosylated IgA1, we foresee that native top-down ECD will become a valuable complementary tool for the de novo sequencing of IgA1s from milk, saliva, or serum.

De novo peptide sequencing using CID and HCD spectra pairs.

In tandem mass spectrometry (MS/MS), there are several different fragmentation techniques possible, including, collision-induced dissociation (CID) higher energy collisional dissociation (HCD), electron-capture dissociation (ECD), and electron transfer dissociation (ETD). When using pairs of spectra for de novo peptide sequencing, the most popular methods are designed for CID (or HCD) and ECD (or ETD) spectra because of the complementarity between them. Less attention has been paid to the use of CID and HCD spectra pairs. In this study, a new de novo peptide sequencing method is proposed for these spectra pairs. This method includes a CID and HCD spectra merging criterion and a parent mass correction step, along with improvements to our previously proposed algorithm for sequencing merged spectra. Three pairs of spectral datasets were used to investigate and compare the performance of the proposed method with other existing methods designed for single spectrum (HCD or CID) sequencing. Experimental results showed that full-length peptide sequencing accuracy was increased significantly by using spectra pairs in the proposed method, with the highest accuracy reaching 81.31%.

Electron capture dissociation in a branched radio-frequency ion trap.

We have developed a high-throughput electron capture dissociation (ECD) device coupled to a quadrupole time-of-flight mass spectrometer using novel branched radio frequency ion trap architecture. With this device, a low-energy electron beam can be injected orthogonally into the analytical ion beam with independent control of both the ion and electron beams. While ions and electrons can interact in a "flow-through" mode, we observed a large enhancement in ECD efficiency by introducing a short ion trapping period at the region of ion and electron beam intersection. This simultaneous trapping mode still provides up to five ECD spectra per second while operating in an information-dependent acquisition workflow. Coupled to liquid chromatography (LC), this LC-ECD workflow provides good sequence coverage for both trypsin and Lys C digests of bovine serum albumin, providing ECD spectra for doubly charged precursor ions with very good efficiency.

ECD of tyrosine phosphorylation in a triple quadrupole mass spectrometer with a radio-frequency-free electromagnetostatic cell.

A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell.

Mechanistic study on electron capture dissociation of the oligosaccharide-Mg²⁺ complex.

Electron capture dissociation (ECD) has shown great potential in structural characterization of glycans. However, our current understanding of the glycan ECD process is inadequate for accurate interpretation of the complex glycan ECD spectra. Here, we present the first comprehensive theoretical investigation on the ECD fragmentation behavior of metal-adducted glycans, using the cellobiose-Mg²⁺ complex as the model system. Molecular dynamics simulation was carried out to determine the typical glycan-Mg²⁺ binding patterns and the lowest-energy conformer identified was used as the initial geometry for density functional theory-based theoretical modeling. It was found that the electron is preferentially captured by Mg²⁺ and the resultant Mg⁺• can abstract a hydroxyl group from the glycan moiety to form a carbon radical. Subsequent radical migration and α-cleavage(s) result in the formation of a variety of product ions. The proposed hydroxyl abstraction mechanism correlates well with the major features in the ECD spectrum of the Mg²⁺-adducted cellohexaose. The mechanism presented here also predicts the presence of secondary, radical-induced fragmentation pathways. These secondary fragment ions could be misinterpreted, leading to erroneous structural determination. The present study highlights an urgent need for continuing investigation of the glycan ECD mechanism, which is imperative for successful development of bioinformatics tools that can take advantage of the rich structural information provided by ECD of metal-adducted glycans.

Mass spectrometric strategies to improve the identification of Pt(II)-modification sites on peptides and proteins.

To further explore the binding chemistry of cisplatin (cis-Pt(NH3)2Cl2) to peptides and also establish mass spectrometry (MS) strategies to quickly assign the platinum-binding sites, a series of peptides with potential cisplatin binding sites (Met(S), His(N), Cys(S), disulfide, carboxyl groups of Asp and Glu, and amine groups of Arg and Lys, were reacted with cisplatin, then analyzed by electron capture dissociation (ECD) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Radical-mediated side-chain losses from the charge-reduced Pt-binding species (such as CH3S(•) or CH3SH from Met, SH(•) from Cys, CO2 from Glu or Asp, and NH2(•) from amine groups) were found to be characteristic indicators for rapid and unambiguous localization of the Pt-binding sites to certain amino acid residues. The method was then successfully applied to interpret the top-down ECD spectrum of an inter-chain Pt-crosslinked insulin dimer, insulin + Pt(NH3)2 + insulin (>10 kDa). In addition, ion mobility MS shows that Pt binds to multiple sites in Substance P, generating multiple conformers, which can be partially localized by collisionally activated dissociation (CAD). Platinum(II) (Pt(II)) was found to coordinate to amine groups of Arg and Lys, but not to disulfide bonds under the conditions used. The coordination of Pt to Arg or Lys appears to arise from the migration of Pt(II) from Met(S) as shown by monitoring the reaction products at different pH values by ECD. No direct binding of cisplatin to amine groups was observed at pH 3 ~ 10 unless Met residues were present in the sequence, but noncovalent interactions between cisplatin hydrolysis and amination [Pt(NH3)4](2+) products and these peptides were found regardless of pH.

Electron capture dissociation of a self-assembled tetranuclear metallosupramolecular complex in the gas phase

A self-assembled tetranuclear metallosupramolecular complex was brought into the gas phase by electrospray ionization (ESI), mass-selected, and fragmented via infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD) in a Fourier-transform ion-cyclotron resonance mass spectrometer (FT-ICR). The resulting spectra were compared. The ECD spectra show some identical fragments to IRMPD which suggest similar fragmentation pathways, but also different fragments which can be assigned to radical cations. Further experiments were performed to investigate the location of the captured electron and results hint at the formation of complexes containing a bipyridyl anion.

Charge Site Mass Spectra: Conformation-Sensitive Components of the Electron Capture Dissociation Spectrum of a Protein

A conventional electron capture dissociation (ECD) spectrum of a protein is uniquely characteristic of the first dimension of its linear structure. This sequence information is indicated by summing the primary c (m+) and z (m+•) products of cleavage at each of its molecular ion's inter-residue bonds. For example, the ECD spectra of ubiquitin (M + nH)(n+) ions, n = 7-13, provide sequence characterization of 72 of its 75 cleavage sites from 1843 ions in seven c ((1-7)+) and eight z ((1-8)+•) spectra and their respective complements. Now we find that each of these c/z spectra is itself composed of "charge site (CS)" spectra, the c (m+) or z (m+•) products of electron capture at a specific protonated basic residue. This charge site has been H-bonded to multiple other residues, producing multiple precursor ion forms; ECD at these residues yields the multiple products of that CS spectrum. Closely similar CS spectra are often formed from a range of charge states of ubiquitin and KIX ions; this indicates a common secondary conformation, but not the conventional α-helicity postulated previously. CS spectra should provide new capabilities for comparing regional conformations of gaseous protein ions and delineating ECD fragmentation pathways.

Glycopeptide Identification Using Liquid-Chromatography-Compatible Hot Electron Capture Dissociation in a Radio-Frequency-Quadrupole Ion Trap

We developed a liquid chromatography (LC) compatible electron capture dissociation (ECD) mass spectrometer for glycoproteomics, with which ECD and hot ECD (HECD) experiments can be flexibly switched by quickly changing the electron energy without further tuning of the mass spectrometer. Desialylated glycopeptides were dissociated well in both ECD and HECD experiments. For sialylated glycopeptides, on the other hand, ECD with electron energy higher than 4 eV showed significantly higher sequence coverage than that with an electron energy of 0.2 eV. A nano LC system was coupled to our ECD mass spectrometer to investigate N-linked glycopeptides from lysylendopeptidase (Lys-C) digests of human transferrin. ECD spectra at multiple electron energies of 0.2, 5.0, and 9.0 eV were obtained for each targeting precursor ion in a single LC injection. Glycopeptides with a sialylated bi-, tri-, or tetra-antennary complex N-glycan were identified with high sequence coverage by HECD. Glycopeptides with tri- or tetra-antennary N-glycans have seldom been analyzed by ECD or ETD before this report. We also found that a preferential dissociation of nonreducing termini of glycans in glycopeptides by ECD and HECD.

Determination of O-Glycosylation Heterogeneity Using a Mass-Spectrometric Method Retaining Sugar Modifications

A mass-spectrometric method for a de novo determination of O-glycosylation heterogeneity was developed. We used a mild fragmentation technique, electron capture dissociation (ECD), which enables the determination of glycosylation sites as well as peptide sequencing. To demonstrate the correct identification of glycopeptides, we prepared a series of glycopeptides with the same peptide sequence and 6 different glycan modifications. ECD spectra were obtained at various electron energies, and were analyzed with the Mascot database-search engine. The obtained candidate glycopeptides were further validated by confirming the spectral overlap of ECD fragment peaks with the theoretical peaks. The results indicate that all glycopeptides were unambiguously identified, including glycosylation sites by combining ECD results with different electron energies for each glycopeptide.

Determination of Peptide Topology through Time-Resolved Double-Resonance under Electron Capture Dissociation Conditions

Characterizing the conformation of biomolecules by mass spectrometry still represents a challenge. With their knotted structure involving a N-terminal macrolactam ring where the C-terminal tail of the peptide is threaded and sterically trapped, lasso peptides constitute an attractive model for developing methods for characterizing gas-phase conformation, through comparison with their unknotted topoisomers. Here, the kinetics of electron capture dissociation (ECD) of a lasso peptide, capistruin, was investigated by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry and compared to that of its branched-cyclic topoisomer, lactam-capistruin. Both peptides produced rather similar ECD spectra but showed different extent of H(•) transfer from c(i)' to z(j)(•) ions. Time-resolved double-resonance experiments under ECD conditions were performed to measure the formation rate constants of typical product ions. Such experiments showed that certain product ions, in particular those related to H(•) transfer, proceeded through long-lived complexes for capistruin, while fast dissociation processes predominated for lactam-capistruin. The formation rate constants of specific ECD product ions enabled a clear differentiation of the lasso and branched-cyclic topoisomers. These results indicate that the formation kinetics of ECD product ions constitute a new way to explore the conformation of biomolecules and distinguish between topoisomers and, more generally, conformers.

Transition Metal Ions: Charge Carriers that Mediate the Electron Capture Dissociation Pathways of Peptides

Electron capture dissociation (ECD) of model peptides adducted with first row divalent transition metal ions, including Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+), were investigated. Model peptides with general sequence of ZGGGXGGGZ were used as probes to unveil the ECD mechanism of metalated peptides, where X is either V or W; and Z is either R or N. Peptides metalated with different divalent transition metal ions were found to generate different ECD tandem mass spectra. ECD spectra of peptides metalated by Mn(2+) and Zn(2+) were similar to those generated by ECD of peptides adducted with alkaline earth metal ions. Series of c-/z-type fragment ions with and without metal ions were observed. ECD of Fe(2+), Co(2+), and Ni(2+) adducted peptides yielded abundant metalated a-/y-type fragment ions; whereas ECD of Cu(2+) adducted peptides generated predominantly metalated b-/y-type fragment ions. From the present experimental results, it was postulated that electronic configuration of metal ions is an important factor in determining the ECD behavior of the metalated peptides. Due presumably to the stability of the electronic configuration, metal ions with fully-filled (i.e., Zn(2+)) and half filled (i.e., Mn(2+)) d-orbitals might not capture the incoming electron. Dissociation of the metal ions adducted peptides would proceed through the usual ECD channel(s) via "hot-hydrogen" or "superbase" intermediates, to form series of c-/z(•)- fragments. For other transition metal ions studied, reduction of the metal ions might occur preferentially. The energy liberated by the metal ion reduction would provide enough internal energy to generate the "slow-heating" type of fragment ions, i.e., metalated a-/y- fragments and metalated b-/y- fragments.

Dissociation Channel Dependence on Peptide Size Observed in Electron Capture Dissociation of Tryptic Peptides

Electron capture dissociation (ECD) of a series of five residue peptides led to the observation that these small peptides did not lead to the formation of the usual c/z ECD fragments, but to a, b, y, and w fragments. In order to determine how general this behavior is for small sized peptides, the effect of peptide size on ECD fragments using a complete set of ECD spectra from the SwedECD spectra database was examined. Analysis of the database shows that b and w fragments are favored for small peptide sizes and that average fragment size shows a linear relationship to parent peptide size for most fragment types. From these data, it appears that most of the w fragments are not secondary fragments of the major z ions, in sharp contrast with the proposed mechanism leading to these ions. These data also show that c fragment distributions depend strongly on the nature of C-terminal residue basic site: arginine leads to loss of short neutral fragments, whereas lysine leads to loss of longer neutral fragments. It also appears that b ions might be produced by two different mechanisms depending on the parent peptide size. A model for the fragmentation pathways in competition is proposed. These relationships between average fragment size and parent peptide size could be further exploited also for CID fragment spectra and could be included in fragmentation prediction algorithms.

Electron Capture, Collision-Induced, and Electron Capture-Collision Induced Dissociation in Q-TOF

Recently, we demonstrated that a radio-frequency-free electromagnetostatic (rf-free EMS) cell could be retrofitted into a triple quad mass spectrometer to allow electron-capture dissociation (ECD) without the aid of cooling gas or phase-specific electron injection into the cell (Voinov et al., Rapid Commun Mass Spectrom 22, 3087-3088, 2008; Voinov et al., Anal Chem 81, 1238-1243, 2009). Subsequently, we used our rf-free EMS cell in the same instrument platform to demonstrate ECD occurring in the same space and at the same time with collision-induced dissociation (CID) to produce golden pairs and even triplets from peptides (Voinov et al., Rapid Commun Mass Spectrom 23, 3028-3030, 2009). In this report, we demonstrate that ECD and CID product-ion mass spectra can be recorded at high resolution with flexible control of fragmentation processes using a newly designed cell installed in a hybrid Q-TOF tandem mass spectrometer.

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminal macrolactam ring. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under collision-induced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure. The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECD spectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD was informative and showed different extent of hydrogen migration (formation of c•/z from c/z•) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-charged MccJ25 and MccJ25-lcm showed a series of radical b-type product ions (b'/•(n)). We proposed that these ions are specific of cyclic-branched peptides and result from a dual c/z• and y/b dissociation, in the ring and in the tail, respectively. This work shows the potentiality of ECD for structural characterization of peptide topoisomers, as well as the effect of conformation on hydrogen migration subsequent to electron capture.

The Generating Function of CID, ETD, and CID/ETD Pairs of Tandem Mass Spectra: Applications to Database Search

Recent emergence of new mass spectrometry techniques (e.g. electron transfer dissociation, ETD) and improved availability of additional proteases (e.g. Lys-N) for protein digestion in high-throughput experiments raised the challenge of designing new algorithms for interpreting the resulting new types of tandem mass (MS/MS) spectra. Traditional MS/MS database search algorithms such as SEQUEST and Mascot were originally designed for collision induced dissociation (CID) of tryptic peptides and are largely based on expert knowledge about fragmentation of tryptic peptides (rather than machine learning techniques) to design CID-specific scoring functions. As a result, the performance of these algorithms is suboptimal for new mass spectrometry technologies or nontryptic peptides. We recently proposed the generating function approach (MS-GF) for CID spectra of tryptic peptides. In this study, we extend MS-GF to automatically derive scoring parameters from a set of annotated MS/MS spectra of any type (e.g. CID, ETD, etc.), and present a new database search tool MS-GFDB based on MS-GF. We show that MS-GFDB outperforms Mascot for ETD spectra or peptides digested with Lys-N. For example, in the case of ETD spectra, the number of tryptic and Lys-N peptides identified by MS-GFDB increased by a factor of 2.7 and 2.6 as compared with Mascot. Moreover, even following a decade of Mascot developments for analyzing CID spectra of tryptic peptides, MS-GFDB (that is not particularly tailored for CID spectra or tryptic peptides) resulted in 28% increase over Mascot in the number of peptide identifications. Finally, we propose a statistical framework for analyzing multiple spectra from the same precursor (e.g. CID/ETD spectral pairs) and assigning p values to peptide-spectrum-spectrum matches.

Top-down high-resolution electron capture dissociation mass spectrometry for comprehensive characterization of post-translational modifications in Rhesus monkey cardiac troponin I

Top-down high-resolution electron capture dissociation (ECD) mass spectrometry (MS) is a powerful approach for comprehensive characterization of large proteins with labile post-translational modifications (PTMs). Phosphorylation of cardiac troponin I (cTnI) is a prominent determinant of cardiac function. Non-human primates (NHPs) are valuable models for biomedical research due to their genetic similarity to humans. To address the emerging need for in-depth understanding of NHP heart models, we have applied top-down high-resolution tandem mass spectrometry in conjugation with immunoaffinity chromatography purification to comprehensively characterize NHP cTnI. Our data revealed that cTnI affinity purified from NHP Rhesus monkey hearts was N-terminally acetylated and mono- or bis-phosphorylated. ECD unambiguously identified Ser22/Ser23 as the only basally phosphorylated sites with a phosphorylation order between these two sites (Ser23 phosphorylates prior to Ser22) which is different from those previously reported for mouse and human cTnI. Our study also strongly supports the nonergodic mechanism of ECD since no neutral loss of phosphates was observed in ECD spectra. Taken together, top-down MS with ECD is extremely valuable for studying labile PTMs in large proteins, which adds critical knowledge to our understanding of protein PTM regulations in health and disease.

Influence of Charge State and Amino Acid Composition on Hydrogen Transfer in Electron Capture Dissociation of Peptides

Although conventional N-Cα bond cleavage in electron capture dissociation (ECD) of multiply-charged peptides generates a complementary c' and z(·) fragment pair, the N-Cα cleavage followed by hydrogen transfer from c' to z(·) fragments produces other fragments, namely c(·) and z'. In this study, the influence of charge state and amino acid composition on hydrogen transfer in ECD is described using sets of peptides. Hydrogen transferred ionic species such as c(·) and z' were observed in ECD spectra of doubly-protonated peptides, while the triply-protonated form did not demonstrate hydrogen transfer. The extent of hydrogen transfer in ECD of doubly-protonated peptides was dependent on constituent amino acids. The ECD of doubly-protonated peptides possessing numerous basic sites showed extensive hydrogen transfer compared with ECD of less basic peptides. The extent of hydrogen transfer is discussed from the viewpoints of the structure of peptide ions, the possibility of internal hydrogen bonding and intermediate lifetime of complex [c' + z(·)].