Electron Capture Dissociation Research Articles

Strategies for Top-Down Hydrogen Deuterium Exchange-Mass Spectrometry: A Mini Review and Perspective.

Hydrogen deuterium-exchange mass spectrometry (HDX-MS) is commonly used in the study of protein dynamics and protein interactions. By measuring the isotopic exchange of backbone amide hydrogens in solution, HDX-MS offers valuable structural insights into challenging biological systems. Traditional HDX-MS approaches utilize bottom-up (BU) proteomics, in which deuterated proteins are digested before MS analysis. BU-HDX enables the characterization of proteins with various sizes in simple protein mixtures or complex biological samples such as cell lysates. However, BU methods are inherently limited by the inability to resolve protein sub-populations arising from different protein conformations, such as those arising from post-translational modifications (PTMs). Alternatively, top-down (TD) HDX-MS detects the global deuterium uptake at the intact proteoform level, allowing direct probing of structural changes due to protein-protein interactions, PTMs, or conformational changes. Combining TD-HDX-MS with electron-based fragmentation techniques, such as electron capture dissociation (ECD) and electron transfer dissociation (ETD), has demonstrated the feasibility of studying intact protein interactions with amino acid-level resolution. Here, we present a brief overview of methodologies, limitations, and applications of TD-HDX-MS using direct infusion techniques and LC-based approaches. Furthermore, we conclude with a perspective on the future directions for TD-HDX-MS.

Advances in Mass Spectrometry Fragmentation Techniques for Peptide and Protein Characterization: CID to ETD

Mass spectrometry (MS) has transformed the field of proteomics by enabling the precise analysis of proteins and peptides at the molecular level. The heart of this analysis is the process of fragmentation, which plays a pivotal role in elucidating amino acid sequence, identifying post-translational modifications (PTMs), and characterizing structural features. Peptides and proteins are first ionized and then subjected to fragmentation techniques such as Collision-Induced Dissociation (CID), Electron-Transfer Dissociation (ETD), and Electron-Capture Dissociation (ECD). These techniques induce fragmentation along the peptide backbone, generating characteristic fragment ions (b and y ions, c and z ions) that reflect the sequence of amino acids. The mass-to-charge (m/z) ratios of these fragment ions provide crucial data for de novo sequencing and PTM identification. Furthermore, fragmentation patterns can reveal insights into protein folding, interactions, and dynamics. This article provides the detailed view of mass fragmentation techniques in peptides and proteins, highlighting its importance in advancing proteomic research and its applications in fields ranging from biomarker discovery to drug development.

Achieving Electron Capture Dissociation in the Radio Frequency Linear Ion Trap without the Assistance of a Magnetic Field─A Simulation Study.

It is extremely difficult to inject a low-energy electron beam into a conventional radiofrequency (RF) linear ion trap for electron capture dissociation (ECD) without using a magnetic field to focus the electrons. In this study, the dynamic process of electrons in an RF field during their injection and transmission through a linear ion trap was simulated to determine the range of the RF phase where the electrons can be decelerated to meet the energy requirement for ECD. The ECD time window was expanded by applying a time-dependent compensation voltage to the cathode. The relationship between the cathode voltage and the phase of the RF voltage was determined. The ECD time window was increased to 49.4% of the total RF cycle after applying a compensation voltage. Between the phases of RF voltage of 0 and 0.975 π, at least 98.7% of electrons can be injected into the ECD reaction zone, and 94% of them had an energy less than 3 eV. The range of electron energy can also easily be shifted upward to enable hot electron capture dissociation.

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

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

Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.

Streamlining the Analysis of Proteins from Snake Venom.

Investigating snake venom is necessary for developing new treatments for envenoming and harnessing the therapeutic potential that lies within venom toxins. Despite considerable efforts in previous research, several technical challenges remain for characterizing the individual components within such complex mixtures. Here, we present native and top-down mass spectrometry (MS) workflows that enable the analysis of individual venom proteins within complex mixtures and showcase the utility of these methodologies on King cobra (Ophiophagus hannah) venom. First, we coupled ion mobility spectrometry for separation and electron capture dissociation for charge reduction to resolve highly convoluted mass spectra containing multiple proteins with masses ranging from 55 to 127 kDa. Next, we performed a top-down glycomic analysis of a 25.5 kDa toxin, showing that this protein contains a fucosylated complex glycan. Finally, temperature-controlled nanoelectrospray mass spectrometry facilitated the top-down sequence analysis of a β-cardiotoxin, which cannot be fragmented by collisional energy due to its disulfide bond pattern. The work presented here demonstrates the applicability of new and promising MS methods for snake venom analysis.

Sequencing of Phosphopeptides Using a Sequential Charge Inversion Ion/Ion Reaction and Electron Capture Dissociation Workflow.

Protein phosphorylation, a common post-translational modification (PTM), is fundamental in a plethora of biological processes, most importantly in modulating cell signaling pathways. Matrix-assisted laser desorption/ionization (MALDI) coupled to tandem mass spectrometry (MS/MS) is an attractive method for phosphopeptide characterization due to its high speed, low limit of detection, and surface sampling capabilities. However, MALDI analysis of phosphopeptides is constrained by relatively low abundances in biological samples and poor relative ionization efficiencies in positive ion mode. Additionally, MALDI tends to produce singly charged ions, generally limiting the accessible MS/MS techniques that can be used for peptide sequencing. For example, collision induced dissociation (CID) is readily amendable to the analysis of singly charged ions, but results in facile loss of phosphoric acid, precluding the localization of the PTM. Electron-based dissociation methods (e.g., electron capture dissociation, ECD) are well suited for PTM localization, but require multiply charged peptide cations to avoid neutralization during ECD. Conversely, phosphopeptides are readily ionized using MALDI in negative ion mode. If the precursor ions are first formed in negative ion mode, a gas-phase charge inversion ion/ion reaction could then be used to transform the phosphopeptide anions produced via MALDI into multiply charged cations that are well-suited for ECD. Herein we demonstrate a multistep workflow combining a charge inversion ion/ion reaction that first transforms MALDI-generated phosphopeptide monoanions into multiply charged cations, and then subjects these multiply charged phosphopeptide cations to ECD for sequence determination and phosphate bond localization.

Negative-Ion Electron Capture Dissociation of MALDI-Generated Peptide Anions.

Negative-ion electron capture dissociation (niECD) is an anion MS/MS technique that provides fragmentation analogous to conventional ECD, including high peptide sequence coverage and retention of labile post-translational modifications (PTMs). niECD has been proposed to be the most efficient for salt-bridged zwitterionic precursor ion structures. Several important PTMs, e.g., sulfation and phosphorylation, are acidic and can, therefore, be challenging to characterize in the positive-ion mode. Furthermore, PTM-friendly techniques, such as ECD, require multiple precursor ion-positive charges. By contrast, singly charged ions, refractory to ECD, are most compatible with niECD. Because electrospray ionization (ESI) typically yields multiply charged ions, we sought to explore matrix-assisted laser desorption/ionization (MALDI) in combination with niECD. However, the requirement for zwitterionic gaseous structures may preclude efficient niECD of MALDI-generated anions. Unexpectedly, we found that niECD of anions from MALDI is not only possible but proceeds with similar or higher efficiency compared with ESI-generated anions. Matrix selection did not appear to have a major effect. With MALDI, niECD is demonstrated up to m/z ∼4300. For such larger analytes, multiple electron captures are observed, resulting in triply charged fragments from singly charged precursor ions. Such charge-increased fragments show improved detectability. Furthermore, significantly improved (∼20-fold signal-to-noise increase) niECD spectral quality is achieved with equivalent sample amounts from MALDI vs ESI. Overall, the reported combination with MALDI significantly boosts the analytical utility of niECD.

New α-Glucosidase Inhibitors from the Whole Plant of Hypericum beanii Based on Ligand Fishing and Molecular Networking Analysis.

In this work, a new rapid and targeted method for screening α-glucosidase inhibitors from Hypericum beanii was developed and verified. Ten new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperlagarol A-J (1-10), and nine known PPAPs (11-19) were obtained from H. beanii. Their structures were identified by using comprehensive analyses involving mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy, and electron capture dissociation calculations. 1 and 2 are two new rare 2,3-seco-spirocyclic PPAPs, 3 and 4 are two novel 12,13-seco-spirocyclic PPAPs, 5 and 6 are two novel spirocyclic PPAPs, 7 and 8 are two new unusual spirocyclic PPAPs with complex bridged ring systems, and 9 and 10 are two novel nonspirocyclic PPAPs. α-GC inhibitory activities of all isolated compounds were tested. Most of them displayed inhibitory activities against α-glucosidase, with the IC50 values ranging from 6.85 ± 0.65 to 112.5 ± 9.03 μM. Moreover, the inhibitory type and mechanism of the active compounds were further analyzed using kinetic studies and molecular docking.

Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of N-Linked Glycopeptides.

Glycosylation is a common modification across living organisms and plays a central role in understanding biological systems and disease. Our ability to probe the gylcome has grown exponentially in the past several decades. However, further improvements to the analytical toolbox available to researchers would allow for increased capabilities to probe structure and function of biological systems and to improve disease treatment. This article applies the developing technique of two-dimensional Fourier transform ion cyclotron resonance mass spectrometry to a glycoproteomic workflow for the standard glycoproteins coral tree lectin (CTL) and bovine ribonuclease B (BRB) to demonstrate its feasibility as a tool for glycoproteomic workflows. 2D infrared multiphoton dissociation and electron capture dissociation spectra of CTL reveal comparable structural information to their 1D counterparts, confirming the site of glycosylation and monosaccharide composition of the glycan. Spectra collected in 2D of BRB reveal correlation lines of fragment ion scans and vertical precursor ion scans for data collected using infrared multiphoton dissociation and diagonal cleavage lines for data collected by electron capture dissociation. The use of similar techniques for glycoproteomic analysis may prove valuable in instances where chromatographic separation is undesirable or quadrupole isolation is insufficient.

Installation protocol for charge transfer dissociation mass spectrometry on ion trapping mass spectrometers.

Charge transfer dissociation (CTD) is a novel fragmentation technique that demonstrates enhanced structural characterization for a wide variety of molecules compared to standard fragmentation techniques like collision-induced dissociation (CID). Alternative fragmentation techniques, such as electron transfer dissociation, electron capture dissociation, and ultraviolet photodissociation, also overcome many of the shortfalls of CID, but none of them are a silver bullet that can adequately characterize a wide variety of structures and charge states of target compounds. Given the diversity of structural classes and their occasional obstinance towards certain activation techniques, alternative fragmentation techniques are required that rely on novel or alternative modes of activation. Herein, we present a step-by-step protocol for the installation of CTD on a quadrupole ion trap mass spectrometer and best practices for optimizing the signal-to-noise ratio and acquisition times for CTD mass spectra. In addition to two CTD installations in the Jackson laboratory, CTD has also been installed, and is currently in operation, on two 3D ion trap mass spectrometers in France: one in the laboratory of Dr. David Ropartz and Dr. Hélène Rogneaux at INRAE in Nantes, and the other in the laboratory of Dr. Jean-Yves Salpin at Université d'Évry Val-d'Essonne, part of the Paris-Saclay University system. Here, we provide a visual protocol to help others accomplish the instrument modification.

Expanding Differential Ion Mobility Separations into the MegaDalton Range.

Along with mass spectrometry (MS), ion mobility separations (IMS) are advancing to ever larger biomolecules. The emergence of electrospray ionization (ESI) and native MS enabled the IMS/MS analyses of proteins up to ∼100 kDa in the 1990s and whole protein complexes and viruses up to ∼10 MDa since the 2000s. Differential IMS (FAIMS) is substantially orthogonal to linear IMS based on absolute mobility K and offers exceptional resolution, unique selectivity, and steady filtering readily compatible with slower analytical methods such as electron capture or transfer dissociation (ECD/ETD). However, the associated MS stages had limited FAIMS to ions with m/z < 8000 and masses under ∼300 kDa. Here, we integrate high-definition FAIMS with the Q-Exactive Orbitrap UHMR mass spectrometer that can handle m/z up to 80,000 and MDa-size ions in the native ESI regime. In the initial evaluation, the oligomers of monoclonal antibody adalimumab (148 kDa) are size-selected up to at least the nonamers (1.34 MDa) with m/z values up to ∼17,000. This demonstrates the survival and efficient separation of noncovalent MDa assemblies in the FAIMS process, opening the door to novel analyses of the heaviest macromolecules.

Salt-Bridged Peptide Anion Gaseous Structures Enable Efficient Negative Ion Electron Capture Dissociation.

We previously discovered that electron attachment to gaseous peptide anions can occur within a relatively narrow electron energy range. The resulting charge-increased radical ions undergo dissociation analogous to conventional cation electron capture/transfer dissociation (ECD/ETD), thus enabling a novel tandem mass spectrometry (MS/MS) technique that we termed negative ion electron capture dissociation (niECD). We proposed that gaseous zwitterionic structures are required for niECD with electron capture either occurring at or being directed by a positively charged site. Here, we further evaluate this zwitterion mechanism by performing niECD of peptides derivatized to alter their ability to form zwitterionic gaseous structures. Introduction of a fixed positive charge tag, a highly basic guanidino group, or a highly acidic sulfonate group to promote zwitterionic structures in singly charged anions, rescued the niECD ability of a peptide refractory to niECD in its unmodified form. We also performed a systematic study of five sets of synthetic peptides with decreasing zwitterion propensity and found that niECD efficiency decreased accordingly, further supporting the zwitterion mechanism. However, traveling-wave ion mobility-mass spectrometry experiments, performed to gain further insight into the gas-phase structures of peptides showing high niECD efficiency, exhibited an inverse correlation between the orientationally averaged collision cross sections and niECD efficiency. These results indicate that compact salt-bridged structures are also a requirement for effective niECD.

Top/Middle-Down Characterization of α-Synuclein Glycoforms.

α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

Characterization of Molecular Tweezer Binding on α-Synuclein with Native Top-Down Mass Spectrometry and Ion Mobility-Mass Spectrometry Reveals a Mechanism for Aggregation Inhibition.

Parkinson's disease, a neurodegenerative disease that affects 15 million people worldwide, is characterized by deposition of α-synuclein into Lewy Bodies in brain neurons. Although this disease is prevalent worldwide, a therapy or cure has yet to be found. Several small compounds have been reported to disrupt fibril formation. Among these compounds is a molecular tweezer known as CLR01 that targets lysine and arginine residues. This study aims to characterize how CLR01 interacts with various proteoforms of α-synuclein and how the structure of α-synuclein is subsequently altered. Native mass spectrometry (nMS) measurements of α-synuclein/CLR01 complexes reveal that multiple CLR01 molecules can bind to α-synuclein proteoforms such as α-synuclein phosphorylated at Ser-129 and α-synuclein bound with copper and manganese ions. The binding of one CLR01 molecule shifts the ability for α-synuclein to bind other ligands. Electron capture dissociation (ECD) with Fourier transform-ion cyclotron resonance (FT-ICR) top-down (TD) mass spectrometry of α-synuclein/CLR01 complexes pinpoints the locations of the modifications on each proteoform and reveals that CLR01 binds to the N-terminal region of α-synuclein. CLR01 binding compacts the gas-phase structure of α-synuclein, as shown by ion mobility-mass spectrometry (IM-MS). These data suggest that when multiple CLR01 molecules bind, the N-terminus of α-synuclein shifts toward a more compact state. This compaction suggests a mechanism for CLR01 halting the formation of oligomers and fibrils involved in many neurodegenerative diseases.

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

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

IM-MS and ECD-MS/MS Provide Insight into Modulation of Amyloid Proteins Self-Assembly by Peptides and Small Molecules.

Neurodegenerative proteinopathies are characterized by formation and deposition of misfolded, aggregated proteins in the nervous system leading to neuronal dysfunction and death. It is widely believed that metastable oligomers of the offending proteins, preceding the fibrillar aggregates found in the tissue, are the proximal neurotoxins. There are currently almost no disease-modifying therapies for these diseases despite an active pipeline of preclinical development and clinical trials for over two decades, largely because studying the metastable oligomers and their interaction with potential therapeutics is notoriously difficult. Mass spectrometry (MS) is a powerful analytical tool for structural investigation of proteins, including protein-protein and protein-ligand interactions. Specific MS tools have been useful in determining the composition and conformation of abnormal protein oligomers involved in proteinopathies and the way they interact with drug candidates. Here, we analyze critically the utilization of ion-mobility spectroscopy-MS (IM-MS) and electron-capture dissociation (ECD) MS/MS for analyzing the oligomerization and conformation of multiple amyloidogenic proteins. We also discuss IM-MS investigation of their interaction with two classes of compounds developed by our group over the last two decades: C-terminal fragments derived from the 42-residue form of amyloid β-protein (Aβ42) and molecular tweezers. Finally, we review the utilization of ECD-MS/MS for elucidating the binding sites of the ligands on multiple proteins. These approaches are readily applicable to future studies addressing similar questions and hold promise for facilitating the development of successful disease-modifying drugs against neurodegenerative proteinopathies.

Digital Quadrupole Isolation and Electron Capture Dissociation on an Extended Mass Range Q-TOF Provides Sequence and Structure Information on Proteins and Protein Complexes.

Electron capture dissociation (ECD) is now a well-established method for sequencing peptides and performing top-down analysis on proteins of less than 30 kDa, and there is growing interest in using this approach for studies of larger proteins and protein complexes. Although much progress on ECD has been made over the past few decades, establishing methods for obtaining informative spectra still poses a significant challenge. Here we describe how digital quadrupole (DigiQ) ion isolation can be used for the mass selection of single charge states of proteins and protein complexes prior to undergoing ECD and/or charge reduction. First, we demonstrate that the DigiQ can isolate single charge states of monomeric proteins such as ubiquitin (8.6 kDa) and charge states of large protein complexes such as pyruvate kinase (234 kDa) using a hybrid quadrupole-TOF-MS (Agilent extended m/z range 6545XT). Next, we demonstrate that fragment ions resulting from ECD can be utilized to provide information about the sequence and structure of the cytochrome c/heme complex and the ubiquitin monomer. Lastly, an especially interesting result for DigiQ isolation and electron capture (EC) was noted; namely, the 16+ charge state of the streptavidin/biotin complex reveals different electron capture patterns for the biotinylated proteoforms of streptavidin. This result is consistent with previous reports that apo streptavidin exists in multiple conformations and that biotin binding shifts the conformational dynamics of the complex (Quintyn, R. Chem. Biol. 2015, 22 (55), 583-592).

Full Separation and Sequencing of Isomeric Proteoforms in the Middle-Down Mass Range Using Cyclic Ion Mobility and Electron Capture Dissociation.

Unambiguous identification of distinct proteoforms and their biological functions is a significant analytical challenge due to the many combinations of post-translational modifications (PTM) that generate isomeric proteoforms. Resulting chimeric tandem mass spectra hinder detailed structural characterization of individual proteoforms for mixtures with more than two isomers. Large isomeric peptides and intact isomeric proteins are extremely difficult to distinguish with traditional chromatographic separation methods. Gas-phase ion separation techniques such as ion mobility spectrometry (IMS) methods now offer high resolving power that may enable separation of isomeric biomolecules, such as peptides and proteins. We explored novel high-resolution cyclic ion mobility spectrometry (cIM) combined with an electro-magnetostatic cell for "on-the-fly" electron capture dissociation (ECD) for separation and sequencing of large isomeric peptides. We demonstrate the effectiveness of this approach on ternary mixtures of mono- and trimethylated isomers of histone H3 N-tails (∼5.4 kDa), achieving a complete separation of these isomers with an average resolving power of ∼400 and a resolution of 1.5 and with nearly 100% amino acid sequence coverage. Our results demonstrate the potential of the cIM-MS/MS(ECD) technology to enhance middle-down and top-down proteomics workflows, thereby facilitating the identification of near-identical proteoforms with essential biological functions in complex mixtures.

Cytotoxicity and Induction of G1 Phase Cell Cycle Arrest in human acute promyelocytic leukemia HL60 cells by indole alkaloids isolated from Dialium corbisieri seeds.

The phytochemical investigation of Dialium corbisieri seeds led to the isolation of five monoterpenoid indole alkaloids along with a phytoserotonin, 1-6 and among the known compounds, the spectroscopic data of (5S)-methoxy-akuammiline (1) was reported for the first time. The structures were elucidated based on NMR spectroscopic techniques such as UV, IR, HRESITOFMS, and ECD spectrum calculations. The isolated compounds were evaluated for their cytotoxicity and cell progression in the human acute promyelocytic leukemia HL60 cell line.