Electron Transfer Dissociation Reaction Research Articles

A broader view on ion heating in traveling-wave devices using fragmentation of CsI clusters and extent of H˙ migration as molecular thermometers.

Electron transfer dissociation (ETD) is becoming increasingly important in mass spectrometry-based analysis of peptides and proteins. Supplemental collisional activation of undissociated electron transfer products can significantly increase fragmentation yield and sequence coverage, but hydrogen rearrangements - specifically, transfer of a hydrogen radical from a c to a z fragment - lead to distorted isotope distributions and increased potential for signal overlap. Concomitant collisional activation during the ion/ion reaction significantly reduces these rearrangements, but, in ion traps, also leads to lower reaction rates and reduced overlap of anion and cation clouds. In traveling-wave ion mobility devices, it has been reported - although not under ETD conditions - that significant ion activation can occur depending on the T-wave height and velocity. Here, we investigate this phenomenon in more detail using a commercial instrument (Waters Synapt G2) and report that a similar effect can be induced within the traveling-wave Trap cell where the ETD reaction occurs, using fairly typical T-wave settings. This ion 'heating' is demonstrated by analyzing the observed isotope distributions (sensitive to the aforementioned hydrogen rearrangements) of ETD fragments of ubiquitin and substance P. A more detailed investigation of ion activation using cesium iodide clusters (without ETD reagent anions present) shows that the observed behavior is consistent with the known dynamics of ions within traveling-wave devices. The insights gained in this work are potentially relevant both for 'native ETD' studies (in which tuning needs to be optimized to avoid unintentional ion activation) as well as the design of future T-wave ETD devices (where this 'heating' effect might be exploited to promote fragment release).

Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation.

Electron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately 3-fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase.

A calibration routine for efficient ETD in large-scale proteomics.

Electron transfer dissociation (ETD) has been broadly adopted and is now available on a variety of commercial mass spectrometers. Unlike collisional activation techniques, optimal performance of ETD requires considerable user knowledge and input. ETD reaction duration is one key parameter that can greatly influence spectral quality and overall experiment outcome. We describe a calibration routine that determines the correct number of reagent anions necessary to reach a defined ETD reaction rate. Implementation of this automated calibration routine on two hybrid Orbitrap platforms illustrate considerable advantages, namely, increased product ion yield with concomitant reduction in scan rates netting up to 75% more unique peptide identifications in a shotgun experiment. Graphical Abstract ᅟ.

Characterisation of glycoproteins using a quadrupole time‐of‐flight mass spectrometer configured for electron transfer dissociation

Electron transfer dissociation (ETD) within ion trapping mass spectrometers has proven to be a useful tool for the characterisation of post-translational modifications. In this study, we describe the implementation of ETD upon a modified quadrupole time-of-flight (Q-ToF) system and methods for the analysis of glycoproteins. Liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS) was performed using a hybrid quadrupole/ion mobility/oa-ToF mass spectrometer equipped with ETD functionality. 1,4-Dicyanobenzene reagent anions necessary for the ETD reaction were generated from a glow discharge region located within the ESI source block. ETD reactions occurred in the stacked ring travelling wave ion guide (located after the quadrupole mass filter and prior to the oa-ToF mass analyser). LC/ETD was performed upon 'super-charged' tryptic glycopeptide ions produced from the recombinant monoclonal antibody trastuzumab. LC/ETD was also performed on ions from the smaller glycopeptides obtained from erythropoietin. ETD performed upon the quadruply 'super-charged' N-linked glycopeptide ions of trastuzumab and the triply charged O-linked glycopeptide ions of erythropoietin provided both glycosylation site assignments and full sequence information, respectively. Tandem mass (MS/MS) spectra employing collision-induced dissociation (CID) were dominated by oxonium product ions hampering full peptide sequence characterisation. LC/ETD on the Q-ToF system proved effective at characterising a number of different N-linked glyco-forms of the tryptic peptide, EEQYNSTYR, from trastuzumab as well as glyco-forms from the O-linked tryptic peptide, EASIPPDAASAAPLR, from erythropoietin. The data demonstrates that the glycopeptide site heterogeneity of trastuzumab and erythropoietin can be accurately characterised. In addition, the post-column mixing of the super-charging reagent, m-NBA, is an effective method to increase the precursor ion charge state and to improve ETD reaction efficiency.

Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.

A novel approach for quantitative peptides analysis by selected electron transfer reaction monitoring

Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) with selective reaction monitoring (SRM) is a selective and sensitive method for quantitation of peptides. SRM is achieved via MS/MS utilizing collision-induced dissociation (CID) while monitoring unique precursor-product ion transitions. Low-energy CID tandem mass spectrometry has been, by far, the most common method used to dissociate peptide ions for sequence analysis. However, collisional scattering of product ions in CID results in decreased intensity of the primary product ion. The lower intensity of the targeted product ion can lead to a reduction in the sensitivity of a quantitative method that uses SRM. Electron transfer dissociation (ETD) is a fragmentation method that is complementary to CID. During the ETD reaction for doubly protonated peptides ([M+2H] 2+), there is a significant shift toward nondissociative electron transfer (ET) product species ([M+2H] + ). We utilized that particular defect in ETD to develop a new quantitative method for monitoring the transition of unique precursors ([M+2H] 2+) to charge-reduced ions ([M+2H] + ). We refer to this method as selective electron transfer reaction monitoring (SETRM). In ESI-MS, trypsin-digested peptides tend to generate doubly protonated peptide precursors. We found that SETRM was more suitable than SRM for these doubly charged tryptic peptides with nano-LC–MS/MS. The quantitative capabilities of SETRM provide a more sensitive way of performing quantitative experiments using the same instrument, thereby improving the application of electron transfer dissociation in proteomics.

Study of Discontinuous Atmospheric Pressure Interfaces for Mass Spectrometry Instrumentation Development

The discontinuous atmospheric pressure interface (DAPI) has allowed the transfer of ions from atmospheric pressure ionization sources to an ion trap mass analyzer in hand-held mass spectrometers with miniature pumping systems at transfer efficiencies high enough for proper chemical analysis. The DAPI potentially would allow a significant enhancement to the mass analysis efficiency of laboratory-scale mass spectrometers, which have pumping systems of much larger capacities. A laboratory-scale mass spectrometer with a DAPI-RIT (rectilinear ion trap)-DAPI configuration has been developed to explore this possibility. The gas dynamic effects on ion trapping and mass analysis have been studied at various conditions. A pulsed nanoelectrospray ionization source synchronized with the DAPI has been implemented to improve the sample usage efficiency as well as to adjust the number of ions to be trapped for MS analysis, so that space charge effects can be avoided. Single-scan spectra of peptides were recorded with an ionization time as short as 1 mus, corresponding to an analyte consumption of several attomoles. The simplicity of application of the DAPI for performing ion/molecule and ion/ion reactions has also been demonstrated with proton transfer and electron transfer dissociation reactions with peptides.

Data Processing Algorithms for Analysis of High Resolution MSMS Spectra of Peptides with Complex Patterns of Posttranslational Modifications

The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) provides the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones. Even with effective gas phase ion isolation so that a single molecular precursor ion is selected, the MSMS spectrum of a heavily modified peptide may reveal the presence of a mixture of peptides with the same amino acid sequence and the same total number of posttranslational modification (PTM) moieties (same PTM composition) but with different PTM configurations or site-specific occupancy isoforms. Currently available data analysis methods depend on a deisotoping procedure, which becomes less effective when spectra (fragmentation patterns) contain many overlapping isotopic distributions. Peptide database search engines can only identify the most abundant PTM configuration (PTM arrangement on different residues) in such mixtures. To identify all the PTM configurations present in these mixtures and to estimate their relative abundances, we extended our fragment assignment by visual assistance program to search for ions representing all possible configurations, subjected to the total PTM composition constraint. This resulted in the identification of PTM configurations supported by unique fragment ions, and their relative abundances were estimated by use of a non-negative least squares procedure.

Data‐dependent electron transfer dissociation of large peptides and medium size proteins in a QTOF instrument on a liquid chromatography timescale

Liquid chromatography (LC) electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) of protein digests is demonstrated in a hybrid quadrupole-hexapole orthogonal time-of-flight (OTOF) mass spectrometer. Analyte ions are selected in a mass-analyzing quadrupole, accumulated in the hexapole linear ETD reaction cell and mutually stored with ETD reagent anions. Product ions are collected in an ion cooler and then analyzed by an OTOF mass analyzer. The hexapole structure of the ETD reaction cell allows for a broad fragment ion mass range distribution and a high ion storage capacity. Analytically useful ETD OTOF-MS/MS spectra could be obtained at a rate of faster than 2 Hz. When used in conjunction with LC this high speed allows for several MS and MS/MS spectra to be obtained across each LC peak. An MS scan is used to select the precursor ions. With a 1 m flight tube and single reflection, resolutions of about 10 k and a mass accuracy of 5 ppm were achieved. When analyzing a 100 fmol solution of a tryptic digest of bovine serum albumin (BSA) by LC/ETD MS/MS, 27 unique peptides were identified with a summed Mascot score of 1316 using the Swiss Prot database. In addition, we explored the capability for analyzing small proteins with the present hybrid instrument. ETD MS/MS of intact ubiquitin ([M+12H](12+)) leads to the identification of the protein with a Mascot score of 264.

A PGC-1α-O-GlcNAc Transferase Complex Regulates FoxO Transcription Factor Activity in Response to Glucose

Metabolic and stress response gene regulation is crucial for the survival of an organism to a changing environment. Three key molecules that sense nutrients and broadly affect gene expression are the FoxO transcription factors, the transcriptional co-activator PGC-1alpha, and the dynamic post-translational modification, O-linked beta-N-acetylglucosamine (O-GlcNAc). Here we identify novel post-translational modifications of PGC-1alpha, including O-GlcNAc, and describe a novel mechanism for how PGC-1alpha co-activates transcription by FoxOs. In liver, in cultured cells, and in vitro with recombinant proteins, PGC-1alpha binds to O-GlcNAc transferase and targets the enzyme to FoxOs, resulting in their increased GlcNAcylation and increased transcriptional activity. Furthermore, glucose-enhanced activation of FoxO1 occurs via this PGC-1alpha-O-GlcNAc transferase-mediated GlcNAcylation. Therefore, one mechanism by which PGC-1alpha can serve as a co-activator of transcription is by targeting the O-GlcNAc transferase to increase GlcNAcylation of specific transcription factors important to nutrient/stress sensing and energy metabolism.

Charger: Combination of Signal Processing and Statistical Learning Algorithms for Precursor Charge-State Determination from Electron-Transfer Dissociation Spectra

Tandem mass spectrometry in combination with liquid chromatography has emerged as a powerful tool for characterization of complex protein mixtures in a high-throughput manner. One of the bioinformatics challenges posed by the mass spectral data analysis is the determination of precursor charge when unit mass resolution is used for detecting fragment ions. The charge-state information is used to filter database sequences before they are correlated to experimental data. In the absence of the accurate charge state, several charge states are assumed. This dramatically increases database search times. To address this problem, we have developed an approach for charge-state determination of peptides from their tandem mass spectra obtained in fragmentations via electron-transfer dissociation (ETD) reactions. Protein analysis by ETD is thought to enhance the range of amino acid sequences that can be analyzed by mass spectrometry-based proteomics. One example is the improved capability to characterize phosphorylated peptides. Our approach to charge-state determination uses a combination of signal processing and statistical machine learning. The signal processing employs correlation and convolution analyses to determine precursor masses and charge states of peptides. We discuss applicability of these methods to spectra of different charge states. We note that in our applications correlation analysis outperforms the convolution in determining peptide charge states. The correlation analysis is best suited for spectra with prevalence of complementary ions. It is highly specific but is dependent on quality of spectra. The linear discriminant analysis (LDA) approach uses a number of other spectral features to predict charge states. We train LDA classifier on a set of manually curated spectral data from a mixture of proteins of known identity. There are over 5000 spectra in the training set. A number of features, pertinent to spectra of peptides obtained via ETD reactions, have been used in the training. The loading coefficients of LDA indicate the relative importance of different features for charge-state determination. We have applied our model to a test data set generated from a mixture of 49 proteins. We search the spectra with and without use of the charge-state determination. The charge-state determination helps to significantly save the database search times. We discuss the cost associated with the possible misclassification of charge states.

Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation.

Glycosylation is an important post-translational modification. Analysis of glycopeptides is difficult using collision-induced dissociation, as it typically yields only information about the glycan structure, without any peptide sequence information. We demonstrate here how a 3D-quadrupole ion trap, using the complementary techniques of collision induced dissociation (CID) and electron-transfer dissociation (ETD), can be used to elucidate the glycan structure and peptide sequence of the N-glycosylated peptide from a fractionated tryptic digest of the lectin from the coral tree, Erythina cristagalli. CID experiments on the multiply protonated glycopeptide ions yield, almost exclusively, cleavage at glycosidic bonds, with little peptide backbone fragmentation. ETD reactions of the triply charged glycopeptide cations with either sulfur dioxide or nitrobenzene anions yield cleavage of the peptide backbone with no loss of the glycan structure. These results show that a 3D-quadrupole ion trap can be used to provide glycopeptide amino acid sequence information as well as information about the glycan structure.