Electronic Excitation Dissociation Research Articles

Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

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.

Electron Impact Excitation of Ions from Organics on Singly Protonated Peptides with and without Post-Translational Modifications.

We report on the dissociation of singly protonated peptides by electrons using electron-activated dissociation (EAD), which comprises electron impact excitation of ions from organics (EIEIO), electronic-excitation dissociation (EED), and electron ionization dissociation (EIoD). Various singly protonated peptides were dissociated using a recently reported fast EAD device. The dissociation can be induced through two pathways: (i) vibrational dissociation similar to collision-activated dissociation (CAD, or collision-induced dissociation, CID) by relaxation from a molecular electronic excited state to high vibrational states; and (ii) radical-induced dissociation where molecular electronic excitation is followed by homolytic cleavage. EAD is complementary to CAD as additional molecular information can be obtained; e.g., fragile PTM moieties, such as glycosylation and sulfation, can be localized. Simultaneously, the energetic production of radical z• fragments enables Leu and Ile discrimination, like in a hot ECD process. Using the fast EAD device, LC-EIEIO-time-of-flight mass spectrometry was applied to a tryptic monoclonal antibody digest containing short singly protonated peptides.

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.

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.

Accurate Identification of Isomeric Glycans by Trapped Ion Mobility Spectrometry-Electronic Excitation Dissociation Tandem Mass Spectrometry.

Ion mobility-mass spectrometry (IM-MS) has become a powerful tool for glycan structural characterization due to its ability to separate isomers and provide collision cross section (CCS) values that facilitate structural assignment. However, IM-based isomer analysis may be complicated by the presence of multiple gas-phase conformations of a single structure that not only increases difficulty in isomer separation but can also introduce the possibility for misinterpretation of conformers as isomers. Here, the ion mobility behavior of several sets of isomeric glycans, analyzed as their permethylated derivatives, in both nonreduced and reduced forms, was investigated by gated-trapped ion mobility spectrometry (G-TIMS). Notably, reducing-end reduction, commonly performed to remove anomerism-induced chromatographic peak splitting, did not eliminate the conformational heterogeneity of permethylated glycans in the gas phase. At a mobility resolving power of ∼100, 14 out of 22 structures showed more than one conformation. These results highlight the need to use IMS devices with high mobility resolving power for better separation of isomers and to acquire additional structural information that can differentiate isomers from conformers. Online electronic excitation dissociation (EED) MS/MS analysis of isomeric glycan mixtures following G-TIMS separation showed that EED can generate isomer-specific fragments while producing nearly identical tandem mass spectra for conformers, thus allowing confident identification of isomers with minimal evidence of any ambiguity resulting from the presence of conformers. G-TIMS EED MS/MS analysis of N-linked glycans released from ovalbumin revealed that several mobility features previously thought to arise from isomeric structures were conformers of a single structure. Finally, analysis of ovalbumin N-glycans from different sources showed that the G-TIMS EED MS/MS approach can accurately determine the batch-to-batch variations in glycosylation profiles at the isomer level, with confident assignment of each isomeric structure.

Variation of Glycosylation Patterns Revealed by PGC‐LC‐ and Gated‐TIMS‐EED Tandem Mass Spectrometry

IntroductionGlycans are the most abundant biopolymers found in nature; they play crucial roles in a wide range of biological activities. Glycan biosynthesis does not follow genetic blueprints, and the resultant glycome is often a mixture of glycoforms with varying compositions, branching patterns, linkages, and stereochemical configurations. Subtle changes in the structure of a glycan can have a profound impact on its biological functions. Recently, we have demonstrated the potential of porous graphitic carbon liquid chromatography electronic excitation dissociation tandem mass spectrometry (PGC‐LC EED MS/MS) and gated‐trapped ion mobility spectrometry (TIMS) negative electron transfer dissociation (NETD) MS/MS in characterizing glycan mixtures, including the separation and identification of isomeric structures. Here, by studying N‐glycans released from different batches of glycoproteins, the glycoform variations at the isomer level were observed. The detailed structure of each isomer could be determined by EED MS/MS and their relative quantification may be achieved by high‐resolution LC or ion mobility separation.MethodsThe released N‐linked glycans were reduced and permethylated before analysis to improve the ionization efficiency and to facilitate spectral interpretation. All experiments were carried out on a 12T solariXTM Fourier‐transform ion cyclotron resonance mass spectrometer equipped with a prototype TIMS device (Bruker Daltonics) or a nanoACQUITY UPLC system (Waters).ResultsRibonuclease B (RNase B) is the glycosylated form of RNase A. With the attachment of a glycan at Asn‐34, the glycosylated ribonuclease showed an increased structural flexibility in the RNA binding region and a much higher activity on double‐stranded RNAs. Previously, application of PGC‐EED MS/MS to the analysis of N‐glycans released from RNase B showed the presence of 18 high‐mannose structures. Here, by comparing the PGC‐LC MS/MS results from two batches of RNase B purchased from different manufacturers, we observed variations in the abundance of each glycoform. Additionally, we have implemented EED MS/MS with gated‐TIMS separation, and showed its ability to resolve and identify isomeric structures. Using gated‐TIMS EED MS/MS, we were able to determine the batch‐to‐batch variation of chicken ovalbumin N‐glycans along with the conformational information.ConclusionsThe PGC‐ and gated‐TIMS‐ EED MS/MS approaches showed advanced performance in separation, characterization, and relative quantification of isomeric glycans, as well as in applications to determine the batch‐to‐batch glycoform variations of glycoproteins. These approaches hold great potential for comprehensive and confident glycan characterization.Support or Funding InformationThis research is supported by the NIH grants P41 GM104603, 703 R21 GM122635, U01CA221234, R01 GM132675, and S10 704 RR025082.

Toward Automatic and Comprehensive Glycan Characterization by Online PGC-LC-EED MS/MS.

Despite the recent advances in mass spectrometry (MS)-based methods for glycan structural analysis, characterization of glycomes remains a significant analytical challenge, in part due to the widespread presence of isomeric structures and the need to define the many structural variables for each glycan. Interpretation of the complex tandem mass spectra of glycans is often laborious and requires substantial expertise. Broad adoption of MS methods for glycomics, within and outside the glycoscience community, has been hindered by the shortage of bioinformatics tools for rapid and accurate glycan sequencing. Here, we developed an online porous graphitic carbon liquid chromatography (PGC-LC)-electronic excitation dissociation (EED) MS/MS method that takes advantage of the superior isomer resolving power of PGC and the structural details provided by EED MS/MS for characterization of glycan mixtures. We also made improvements to GlycoDeNovo, our de novo glycan sequencing algorithm, so that it can automatically and accurately identify glycan topologies from EED tandem mass spectra acquired online. The majority of linkages can also be determined de novo, although in some cases, biological insight may be needed to fully define the glycan structure. Application of this method to the analysis of N-glycans released from ribonuclease B not only revealed the presence of 18 high-mannose structures, including new isomers not previously reported, but also provided relative quantification for each isomeric structure. With fully automated data acquisition and topology analysis, the approach presented here holds great potential for automated and comprehensive glycan characterization.

Characterization of Isomeric Glycans by Reversed Phase Liquid Chromatography-Electronic Excitation Dissociation Tandem Mass Spectrometry.

The occurrence of numerous structural isomers in glycans from biological sources presents a severe challenge for structural glycomics. The subtle differences among isomeric structures demand analytical methods that can provide structural details while working efficiently with on-line glycan separation methods. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool for mixture analysis, the commonly utilized collision-induced dissociation (CID) method often does not generate a sufficient number of fragments at the MS2 level for comprehensive structural characterization. Here, we studied the electronic excitation dissociation (EED) behaviors of metal-adducted, permethylated glycans, and identified key spectral features that could facilitate both topology and linkage determinations. We developed an EED-based, nanoscale, reversed phase (RP)LC-MS/MS platform, and demonstrated its ability to achieve complete structural elucidation of up to five structural isomers in a single LC-MS/MS analysis. Graphical Abstract.

De Novo Glycan Sequencing by Electronic Excitation Dissociation and Fixed-Charge Derivatization.

Detailed glycan structural characterization is frequently achieved by collisionally activated dissociation (CAD) based sequential tandem mass spectrometry (MS n) analysis of permethylated glycans. However, it is challenging to implement MS n ( n > 2) during online glycan separation, and this has limited its application to analysis of complex glycan mixtures from biological samples. Further, permethylation can reduce liquid chromatographic (LC) resolution of isomeric glycans. Here, we studied the electronic excitation dissociation (EED) fragmentation behavior of native glycans with a reducing-end fixed charge tag and identified key spectral features that are useful for topology and linkage determination. We also developed a de novo glycan sequencing software that showed remarkable accuracy in glycan topology elucidation based on the EED spectra of fixed charge-derivatized glycans. The ability to obtain glycan structural details at the MS2 level, without permethylation, via a combination of fixed charge derivatization, EED, and de novo spectral interpretation, makes the present approach a promising tool for comprehensive and rapid characterization of glycan mixtures.

Asparagine-Linked Glycans of Cryptosporidium parvum Contain a Single Long Arm, Are Barely Processed in the Endoplasmic Reticulum (ER) or Golgi, and Show a Strong Bias for Sites with Threonine

Cryptosporidium parvum causes severe diarrhea in infants in developing countries and in immunosuppressed persons, including those with AIDS. We are interested in the Asn-linked glycans (N-glycans) of C. parvum, because (1) the N-glycan precursor is predicted to contain five mannose and two glucose residues on a single long arm versus nine mannose and three glucose residues on the three-armed structure common in host N-glycans, (2) C. parvum is a rare eukaryote that lacks the machinery for N-glycan-dependent quality control of protein folding in the lumen of the Endoplasmic Reticulum (ER), and (3) ER and Golgi mannosidases, as well as glycosyltransferases that build complex N-glycans, are absent from the predicted proteome. The C. parvum N-glycans reported here, which were determined using a combination of collision-induced dissociation and electronic excitation dissociation, contain a single, unprocessed mannose arm ± terminal glucose on the trimannosyl chitobiose core. Upon nanoUPLC-MS/MS separation and analysis of the C. parvum tryptic peptides, the total ion and extracted oxonium ion chromatograms delineated 32 peptides with occupied N-glycan sites; these were derived from 16 glycoproteins. Although the number of potential N-glycan sites with Thr (NxT) is only about twice that with Ser (NxS), almost 90% of the occupied N-glycan sites contain NxT. The two most abundant C. parvum proteins modified with N-glycans were an immunodominant antigen on the surface of sporozoites (gp900) and the possible oocyst wall protein 1 (POWP1). Seven other glycoproteins with N-glycans were unique to C. parvum; five shared common ancestry with other apicomplexans; two glycoproteins shared common ancestry with many organisms. In summary, C. parvum N-glycans are remarkable for the absence of ER and Golgi modification and for the strong bias toward occupancy of N-glycan motifs containing Thr.

Mechanistic Study on Electronic Excitation Dissociation of the Cellobiose-Na(+) Complex.

The recent development of electron activated dissociation (ExD) techniques has opened the door for high-throughput, detailed glycan structural elucidation. Among them, ExD methods employing higher-energy electrons offer several advantages over low-energy electron capture dissociation (ECD), owing to their applicability towards chromophore-labeled glycans and singly charged ions, and ability to provide more extensive structural information. However, a lack of understanding of these processes has hindered rational optimization of the experimental conditions for more efficient fragmentation as well as the development of informatics tools for interpretation of the complex glycan ExD spectra. Here, cellobiose-Na(+) was used as the model system to investigate the fragmentation behavior of metal-adducted glycans under irradiation of electrons with energy exceeding their ionization potential, and served as the basis on which a novel electronic excitation dissociation (EED) mechanism was proposed. It was found that ionization of the glycan produces a mixture of radical cations and ring-opened distonic ions. These distonic ions then capture a low-energy electron to produce diradicals with trivial singlet-triplet splitting, and subsequently undergo radical-induced dissociation to produce a variety of fragment ions, the abundances of which are influenced by the stability of the distonic ions from which they originate. Graphical Abstract ᅟ.

Detailed Glycan Structural Characterization by Electronic Excitation Dissociation

The structural complexity and diversity of glycans parallel their multilateral functions in living systems. To better understand the vital roles glycans play in biological processes, it is imperative to develop analytical tools that can provide detailed glycan structural information. This was conventionally achieved by multistage tandem mass spectrometry (MS(n)) analysis using collision-induced dissociation (CID) as the fragmentation method. However, the MS(n) approach lacks the sensitivity and throughput needed to analyze complex glycan mixtures from biological sources, often available in limited quantities. We define herein the critical parameters for a recently developed fragmentation technique, electronic excitation dissociation (EED), which can yield rich structurally informative fragment ions during liquid chromatographic (LC)-MS/MS analysis of glycans. We further demonstrate that permethylation, reducing end labeling and judicious selection of the metal charge carrier, can greatly facilitate spectral interpretation. With its high sensitivity, throughput, and compatibility with online chromatographic separation techniques, EED appears to hold great promise for large-scale glycomics studies.

Energy-Dependent Electron Activated Dissociation of Metal-Adducted Permethylated Oligosaccharides

The effects of varying the electron energy and cationizing agents on electron activated dissociation (ExD) of metal-adducted oligosaccharides were explored, using permethylated maltoheptaose as the model system. Across the examined range of electron energy, the metal-adducted oligosaccharide exhibited several fragmentation processes, including electron capture dissociation (ECD) at low energies, hot-ECD at intermediate energies, and electronic excitation dissociation (EED) at high energies. The dissociation threshold depended on the metal charge carrier(s), whereas the types and sequence spans of product ions were influenced by the metal-oligosaccharide binding pattern. Theoretical modeling contributed insight into the metal-dependent behavior of carbohydrates during low-energy ECD. When ExD was applied to a permethylated high mannose N-linked glycan, EED provided more structural information than either collision-induced dissociation (CID) or low-energy ECD, thus demonstrating its potential for oligosaccharide linkage analysis.

Gas-phase synthesis and reactivity of binuclear gold hydride cations, (R3PAu)2H+ (R = Me and Ph)

Electrospray ionization of a mixture of the two gold phosphine chlorides, R3PAuCl (R = Ph and Me), silver nitrate and the amino acid N,N-dimethylglycine (DMG) yields a range of gold containing cluster ions including: (R3P)Au(PR'3)+; (R3PAu)(R'3PAu)Cl+ and (R3PAu)(R'3PAu)(DMG-H)+ (where R = R' = Ph; R = R' = Me; R = Me and R' = Ph). Collision induced dissociation (CID) of the (R3PAu)(R'3PAu)(DMG-H)+ precursor ions yielded the hitherto unknown gold hydride dimers (R3PAu)(R'3PAu)H+. The gas-phase chemistry of these dimers was studied using ion-molecule reactions, collision induced dissociation, electronic excitation dissociation (EED) and DFT calculations on the (H3PAu)2H+ model system. A novel phosphine ligand migration was found to occur prior to fragmentation under CID conditions and this was supported by DFT calculations, which revealed a transition state with a bridging phosphine ligand.

The role of electron capture dissociation in biomolecular analysis.

The introduction of electron capture dissociation (ECD) to electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) constitutes a significant advance in the structural analysis of biomolecules. The fundamental features and benefits of ECD are discussed in this review. ECD is currently unique to FT-ICR MS and the fundamentals of that technique are outlined. The advantages and complementarity of ECD in relation to other tandem mass spectrometry (MS/MS) techniques, such as infrared multiphoton dissociation (IRMPD) and sustained off-resonance collision-induced dissociation (SORI-CID), are discussed. The instrumental considerations associated with implementation of ECD, including activated ion techniques and coupling to on-line separation techniques, are covered, as are the allied processes electronic excitation dissociation (EED), electron detachment dissociation (EDD), and hot electron capture (HECD). A major theme of this review is the role of ECD in proteomics, particularly for characterization of post-translational modifications (phosphorylation, glycosylation, carboxyglutamic acid, sulfation, acylation, and methionine oxidation) and the top-down approach to protein identification. The application of ECD to the analysis of polymers, peptide nucleic acids, and oligonucleotides is also discussed.

Reactions of Polypeptide Ions with Electrons in the Gas Phase

AbstractFor Abstract see ChemInform Abstract in Full Text.

Tandem MALDI/EI ionization for tandem Fourier transform ion cyclotron resonance mass spectrometry of polypeptides

The tandem ionization (TI) technique in which protonated polypeptides [M+H] + produced by matrix-assisted laser desorption ionization (MALDI) are further ionized inside a Fourier transform (FT) mass spectrometer by >10 eV electrons has been extended to allow the formed radical dications [M+H] 2+ to capture low energy electrons. The capture produces [M+H] +∗ ions which, being isoelectronic with the precursor ions, are electronically excited. The electron capture process is >10 eV exothermic and may cause fragmentation, mainly into even-electron a and c′ ions. This fragmentation bears similarity with UV photodissociation, but produces little secondary fragmentation. Up to date, 10 peptides from 1 to 3 kDa have been subjected to this electronic excitation dissociation (EED). The details and potential analytical applications of EED are discussed.

Intramolecular hydrogen atom transfer in hydrogen-deficient polypeptide radical cations

Irradiation of protonated polypeptides NH 2–RH +–COOH by >10 eV electrons leads to further ionization and fast intramolecular charge transfer to the free N-terminus. The resulting species may undergo further hydrogen atom rearrangement to form distonic ions N +H 3–RH +–COO . Such transfer is exothermic but can involve an appreciable barrier, e.g., 2.3±0.5 eV for MH 2+ ions of the peptide ACTH 1–10. Radical polypeptide dications can, therefore, be viewed as hydrogen atom wires. Subsequent capture of low energy electrons results in fragmentation. The pattern of this electronic excitation dissociation (EED) is consistent with hydrogen transfer prior to electron capture.