Metastable Fragmentation Research Articles

A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines.

Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable. Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30. A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker. Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates.

Time-Resolved Imaging of High Mass Proteins and Metastable Fragments Using Matrix-Assisted Laser Desorption/Ionization, Axial Time-of-Flight Mass Spectrometry, and TPX3CAM.

The Timepix (TPX) is a position- and time-sensitive pixelated charge detector that can be coupled with time-of-flight mass spectrometry (TOF MS) in combination with microchannel plates (MCPs) for the spatially and temporally resolved detection of biomolecules. Earlier generation TPX detectors used in previous studies were limited by a moderate time resolution (at best 10 ns) and single-stop detection for each pixel that hampered the detection of ions with high mass-to-charge (m/z) values at high pixel occupancies. In this study, we have coupled an MCP-phosphor screen-TPX3CAM detection assembly that contains a silicon-coated TPX3 chip to a matrix-assisted laser desorption/ionization (MALDI)-axial TOF MS. A time resolution of 1.5625 ns, per-pixel multihit functionality, simultaneous measurement of TOF and time-over-threshold (TOT) values, and kHz readout rates of the TPX3 extended the m/z detection range of the TPX detector family. The detection of singly charged intact Immunoglobulin M ions of m/z value approaching 1 × 106 Da has been demonstrated. We also discuss the utilization of additional information on impact coordinates and TOT provided by the TPX3 compared to conventional MS detectors for the enhancement of the quality of the mass spectrum in terms of signal-to-noise (S/N) ratio. We show how the reduced dead time and event-based readout in TPX3 compared to the TPX improves the sensitivity of high m/z detection in both low and high mass measurements (m/z range: 757-970,000 Da). We further exploit the imaging capabilities of the TPX3 detector for the spatial and temporal separation of neutral fragments generated by metastable decay at different locations along the field-free flight region by simultaneous application of deflection and retarding fields.

Mass Spectrometry of Au10(4-tert-butylbenzenethiolate)10 Nanoclusters Using Superconducting Tunnel Junction Cryodetection Reveals Distinct Metastable Fragmentation.

Cryodetection mass spectrometry (MS) was used to study the Au10(TBBT)10 (TBBT = 4-tert-butylbenzenethiolate) catenane nanocluster. The matrix-assisted laser desorption ionization (MALDI) process generates distinct fragments that can be arranged into two distinct regimes: (i) in-source fragmentation, which occurs rapidly in a relatively short (<170 ns) time frame, and (ii) metastable fragmentation, which occurs postacceleration during a time-of-flight (TOF) mass analysis over a longer time frame (>170 ns-250 μs). Using MALDI-TOF MS with superconducting tunnel junction (STJ) cryodetection, distinct metastable nanocluster fragments were resolved at lower energies deposited into the detector. The results also demonstrated that STJ cryodetection MS can be used to acquire multiple (>10), simultaneous tandem mass spectra in a single experiment. Simulated fragmentation of the Au10 nanocluster using ab initio molecular dynamics (AIMD) revealed the different fragmentation processes and confirmed the MS results. Using both the empirical MS data and AIMD calculations, fragmentation pathways are proposed for Au10(TBBT)10, which terminate with two small, stable ringed species.

Microsecond dynamics of molecular negative ions formed by low-energy electron attachment to fluorinated tetracyanoquinodimethane.

Low-energy (0-15eV) electron interactions with gas-phase 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4-TCNQ) molecules are studied under single collision conditions using dissociative electron attachment spectroscopy. The experimental findings are supported by density functional theory calculations of the virtual orbital energies and energetics of the dissociative decays. Long-lived molecular negative ions F4-TCNQ- are detected in a wide electron energy range (0-3eV) with electron detachment times in the range of milliseconds. Although plenty of decay channels are observed, their intensities are found to be very small (two to four orders of magnitude relative to the F4-TCNQ- signal). These findings prove that the structure of this strong electron-accepting molecule bearing an excess electron is robust in its electronic ground state, even when highly (up to 6eV) vibrationally excited. As many as nine metastable fragment anions formed slowly (in the 16-23 µs range) are found in the negative ion mass spectrum of F4-TCNQ, as never observed before in compounds possessing high electron-accepting ability. The present results shed some light on microsecond dynamics of isolated F4-TCNQ molecules under conditions of excess negative charge, which are important for understanding the functionality of nanoscale devices containing this molecule as a structural element.

Dissociative electron attachment to fluorinated nitrobenzenes

Low-energy electron attachment to three fluorinated nitrobenzene molecules in the gas phase in the energy range from zero to 10 eV is reported. Non-dissociative and dissociative electron attachment to 3,5-difluoronitrobenzene, 2,3,4-trifluoronitrobenzene and 2,4,6-trifluoronitrobenzene were observed. The experiments were carried out utilizing a trochoidal electron monochromator coupled to a time-of-flight mass spectrometer. Quantum chemical calculations of the major dissociation pathways have been made to compare thermochemical reaction balances with the experimental findings. The most dominant fragmentation channels involve (partial) dissociation of the nitro group, which is influenced by the fluorination of the molecule. In addition to prompt dissociation in the interaction region, slower metastable fragmentation of the nitro groups via loss of a neutral NO was also observed on microsecond timescales. Remarkably, we also observe the formation of the parent anion of 3,5-difluoronitrobenzene at non-thermal energies.

Identification of Antibacterial Immunity Proteins in &lt;em&gt;Escherichia coli&lt;/em&gt; using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation.

The endoplasmic reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins. However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use a substrate trapping approach in combination with quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosaccharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4-dependent ERAD pathway. RHBDL4-catalysed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. RHBDL4 thereby controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning.

Mapping the complex metastable fragmentation pathways of excited 3-aminophenol+

This work applies the technique of mapping ion detection using a reflectron mass spectrometer against flight-time and reflection voltage to elucidate the complex metastable fragmentation pattern of the biomolecular ion 3-aminophenol+ (3-AP+, C6H7NO+). Multi-photon ionization experiments revealed the excited ion's fragmentation routes for the first time and comparisons with calculated flight-times enabled 18 different μs-timescale dissociations to be assigned. These included the rare observation of a double hydrogen loss channel from a fragment ion. DFT calculations provided further insights into the most prominent apparent fragmentation sequence: 3-AP+ (m/z 109) → HCO + C5H6N+ (m/z 80) → CNH + C4H5+ (m/z 53) → C2H2 + C2H3+ (m/z 27).

Characterization of Mega-Dalton-Sized Nanoparticles by Superconducting Tunnel Junction Cryodetection Mass Spectrometry.

The characterization of nanomaterials is critical to understand the size/structure-dependent properties of these particles. In this report, a form of heavy ion mass spectrometry, namely, superconducting tunnel junction (STJ) cryodetection mass spectrometry (MS) is used to characterize quantum dot semiconductor nanocrystals and gold nanoparticles. The nanoparticles studied ranged in mass from 200 kDa to >1.5 MDa and included lead sulfide quantum dots, various cadmium selenide and/or telluride-based core-shell quantum dots coated with different ligands, and gold nanoparticles. Nanoparticles were ionized by both matrix-assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI), shot with an aimed ion gun into a flight tube, mass separated by time-of-flight (TOF), and detected by an energy-sensitive STJ cryodetector. STJ cryodetection MS can be used to analyze intact heterogeneous nanoparticles, allowing determination of average particle mass, dispersity, and ligand loading. Some nanoparticles, however, do undergo fragmentation during the MALDI or LDI-TOF mass analyses. The measurement of the energy deposited into the detector was found to be different for different types of particles. Metastable fragments from these nanoparticles were observed at lower energies. The lower energies deposited for metastable fragments can provide insight into the stability and surface compositions of these materials. Cadmium selenide core-shell quantum dots (655 nm emission) conjugated to biomacromolecules, such as cholera toxin B and human serum transferrin, were also analyzed. When compared to unconjugated particles by mass, it was determined that ∼96 cholera toxin B and ∼14 transferrin proteins were attached to the surface of these nanoparticles.

Characterization of ZnO Nanoparticles using Superconducting Tunnel Junction Cryodetection Mass Spectrometry.

Zinc oxide (ZnO) nanoparticles coated with either n-octylamine (OA) or α-amino poly(styrene-co-acrylonitrile) (PSAN) ligands (L) have been analyzed using laser desorption/ionization and matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) superconducting tunnel junction (STJ) cryodetection mass spectrometry. STJ cryodetection has the advantage of high m/z detection and allows for the determination of average molecular weights and dispersities for 500-600 kDa ZnO-L nanoparticles. The ability to detect the relative energies deposited into the STJs has allowed for investigation of ZnO-L metastable fragmentation. ZnO-L precursor ions gain enough internal energy during the MALDI process to undergo metastable fragmentation in the flight tube. These fragments produced a lower energy peak, which was assigned as ligand-stripped ZnO cores whereas the individual ligands were at too low of an energy to be observed. From these STJ energy resolved peaks, the average weight percentage of inorganic material making up the nanoparticle was determined, where ZnO-OA and ZnO-PSAN nanoparticles are comprised of ~62% and ~68% wt ZnO, respectively. In one example, grafting densities were calculated based on the metastable fragmentation of ligands from the core to be 16 and 1.1 nm-2 for ZnO-OA and ZnO-PSAN, respectively, and compared with values determined by thermogravimetric analysis (TGA) and transmission electron microscopy (TEM). Graphical Abstract ᅟ.

Insights on Clusters Formation Mechanism by Time of Flight Mass Spectrometry. 2. The Case of Acetone-Water Clusters.

This paper is the second of a series dealing with clusters formation mechanism. In part 1, water clusters with the addition of an electrophilic molecule such as ethanol were studied by Time Of Flight Mass Spectrometry (TOFMS). Mass distributions of molecular clusters of ethanol, water and ethanol-water mixed clusters, were obtained by means of two different ionization methods: Electron Ionization (EI) and picosecond laser Photo-Ionization (PI) at a wavelength of 355nm. In part 2, the same experimental approach was employed to obtain mass spectra of clusters generated by acetone-water binary mixtures with a different composition. Strong dependence of the mass spectra of clusters with EI and PI on the acetone-water mixing ratio was observed. It was shown that the spectral pattern changes gradually and water-rich cluster signals become fainter while acetone-rich cluster signals become more intensive with increasing acetone concentrations from 0.3% to 40%. Owing to the hydrogen bond acceptor character of acetone, its self-association is discouraged with respect to ethanol. The autocorrelation function (AF) was used to analyze the variation of the water clusters composition with the increase of the acetone concentration in terms of fundamental periodicities. However, although acetone and ethanol present a very different hydrogen-bonding ability, similarly to ethanol-water system, in acetone-water system the formation of water-rich clusters and subsequent metastable fragmentation are the dominant process that determine the clusters distribution, irrespective of the ionization process, while the ionization process significantly affects the acetone-rich clusters distribution. Graphical Abstract ᅟ.

Thermal radiation and fragmentation pathways of photo-excited silicon clusters.

The fragmentation of laser heated silicon clusters was studied by time-of-flight mass spectrometry. For Si(n)(+) (n = 5-19, 21), the lowest energy fragmentation pathways were identified as the metastable decay channel occurring after the primary acceleration of the ions. The radiative cooling of laser excited Si(n)(+) (n = 5-9, 11, and 13) was quantified via its quenching effect on the amount of metastable fragmentation. The quenching varied strongly with cluster size, from no observable amount for Si7(+) to a cooling constant of 3 ⋅ 10(5) s(-1) for Si13(+). In addition, based on the observed fragmentation channels, the ionization energies and the relative binding energies of the clusters were partially ordered, and several ionization energies have been bracketed more precisely.

Metastable decay of nitrogen clusters ions, and determination of the average kinetic energy release and binding energy values

We have carried out measurements on metastable fragmentation of mass selected nitrogen cluster ions, (N2)n+, which are produced by electron impact ionization of a neutral nitrogen cluster beam. We have derived binding energies for n=5–10, from the kinetic energy released in metastable decay reactions using finite heat bath theory. Our results are compared with other experimental results and with quantum chemical calculations.

Insights on Clusters Formation Mechanism by Time of Flight Mass Spectrometry. 1. The Case of Ethanol-Water Clusters.

In the present work, water clusters with the addition of an electrophilic molecule such as ethanol have been studied by time of flight mass spectrometry (TOFMS). Mass distributions of molecular clusters of ethanol, water, and ethanol-water mixed clusters were obtained by two different ionization methods: electron ionization (EI) and picosecond laser photo-ionization (PI) at a wavelength of 355 nm. It was shown that short pulse laser ionization increases the signal intensity and promotes the extension of the detected mass range of the clusters in comparison with EI. Much larger clusters were detected in our experiments with respect to the current literature. The autocorrelation function (AF) was introduced in the analysis of the composition of the water clusters in terms of fundamental periodicities for obtaining information on clusters formation mechanisms. Besides, it was found that ethanol molecules are capable of substitutional interaction with hydrogen-bonded water clusters in ethanol-water binary mixtures but the self-association of ethanol was the dominant process. Moreover, the increase of ethanol concentration promotes both the formation of hydrated ethanol clusters and the self-association of ethanol clusters in ethanol-water binary mixtures. The formation of water-rich clusters and subsequent metastable fragmentation were found to be the dominant processes determining the water-rich cluster distribution, irrespective of the ionization process, while the ionization process significantly affects the ethanol-rich cluster distribution.

Proton-Shuttle-Assisted Heterolytic Carbon–Carbon Bond Cleavage and Formation

The conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP) catalyzed by DXP reductoisomerase (DXR) is the committing step in the biosynthesis of terpenoids. This MEP pathway is essential for most pathogenic bacteria but absent in human, and thus, it is an attractive target for the development of novel antibiotics. To this end, it is critical to elucidate the conversion mechanism and identify the transition state, as many drugs are transition-state analogues. Here we performed extensive combined quantum mechanical (density functional theory B3LYP/6-31G*) and molecular mechanical molecular dynamics simulations to elucidate the catalytic mechanism. Computations confirmed the transient existence of two metastable fragments of DXP by the heterolytic C3–C4 bond cleavage, namely, 1-propene-1,2-diol and glycoaldehyde phosphate, in accord with the most recent kinetic isotope effect (KIE) experiments. Significantly, the heterolytic C3–C4 bond cleavage and C2–C4 bond formation are accompanied by proton shuttles, which significantly lower their reaction barriers to only 8.2–6.0 kcal/mol, compared with the normal single carbon–carbon bond energy 83 kcal/mol. This mechanism thus opens a novel way for the design of catalysts in the cleavage or formation of aliphatic carbon–carbon bonds.

Metastable fragment production at the C 1s and O 1s edges of the CO2 molecule

We have studied the dissociation of the CO2 molecule into neutral fragments by observing coincidences between neutral metastable fragments and photoions at photon energies between 250 eV and 600 eV. We attribute the metastable signal to oxygen atoms in the 2s22p3(4S)3s(5S) state and to fragments in high Rydberg states. Neutral metastable fragments are produced by the dissociation of valence-excited states of that are populated by direct photoionization or, more efficiently, by core excitation and ionization followed by Auger decay. Metastable fragments were detected at all C 1s and O 1s single excitations, and their intensity was enhanced particularly just above the O 1s and C 1s ionization thresholds, where the recapture of slow photoelectrons is possible. They were also abundantly created at core-valence double excitations, at the thresholds of O 1s and C 1s in shake-up satellites, and to a lesser extent, also in regions of the photoionization continua where there are no resonances.

Multiphoton ionization of large water clusters.

Water clusters are multimers of water molecules held together by hydrogen bonds. In the present work, multiphoton ionization in the UV range coupled with time of flight mass spectrometry has been applied to water clusters with up to 160 molecules in order to obtain information on the electronic states of clusters of different sizes up to dimensions that can approximate the bulk phase. The dependence of ion intensities of water clusters and their metastable fragments produced by laser ionization at 355 nm on laser power density indicates a (3+1)-photon resonance-enhanced multiphoton ionization process. It also explains the large increase of ionization efficiency at 355 nm compared to that at 266 nm. Indeed, it was found, by applying both nanosecond and picosecond laser ionization with the two different UV wavelengths, that no water cluster sequences after n = 9 could be observed at 266 nm, whereas water clusters up to m/z 2000 Th in reflectron mode and m/z 3000 Th in linear mode were detected at 355 nm. The agreement between our findings on clusters of water, especially true in the range with n > 10, and reported data for liquid water supports the hypothesis that clusters above a critical dimension can approximate the liquid phase. It should thus be possible to study clusters just above 10 water molecules, for getting information on the bulk phase structure.

A revisit of high collision energy effects on collision-induced dissociation spectra using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-LIFT-TOF/TOF): application to the sequencing of RNA/DNA chimeras.

High-energy collision-induced dissociation (CID) spectra of isomeric RNA/DNA chimeras using matrix-assisted laser desorption/ionization time-of-flight LIFT mass spectrometry (MALDI-LIFT-TOF/TOF) can potentially be applied for an exhaustive fragment characterization in a nucleic acid sequencing scheme. These chimeras contain deoxynucleotides and at the 3'-end a ribonucleotide with a 3'-phosphate group. Deprotonated RNA/DNA chimeras of 4-, 5-, 7- and 10-mers are analyzed by CID. This enhances consecutive dissociations from both the precursor and prompt product anions generated by MALDI and metastable fragmentations prior to entering the LIFT cell. Gas-phase fragmentations of 4- and 5-mers produced many fragment ions, from base release prior to consecutive cleavage of the nucleotide phosphate bond linkage phosphate. The unusual a4(-) product ion is a specific and diagnostic dissociation of the 4-mer if the ribonucleotide contains cytosine. As the size of RNA/DNA chimeras increase, several abundant product ions are generated mainly from zwitterionic forms (deprotonated phosphate ester and protonated base sites): [(M-H)-BiH](-), [ai-BiH](-), wj(-), [wj, (ai-BiH)](-) (if Bi ≠ T) as internal product ion, and more rarely [wj-BiH](-). The absence of the majority of the [ai-BiH](-) series although the wj (-) series suggested that the higher critical energy processes with a loose transition state are favored yielding the wj(-) series. A large number of abundant fragment ions are detected which enable each isomer to be sequenced. This sequencing method is high-throughput, accurate and could be used to sequence isomers of up to 10-mers and also oligonucleotides of unknown sequence. However, RNA/DNA chimeras without thymine must be sufficiently concentrated to reach desorption of deprotonated molecular species to be selected in LIFT to produce all fragment ions within measurable abundances.

Suppression of unimolecular decay of laser desorbed peptide and protein ions by entrainment in rarefied supersonic gas jets under weak electric fields

Unimolecular decay of sample ions imposes a limit on the usable laser fluence in matrix-assisted laser desorption/ionization (MALDI) ion sources. Traditionally, some modest degree of collisional sample ion cooling has been achieved by connecting MALDI ion sources directly to gas-filled radio frequency (RF) multipoles. It was also discovered in the early 1990s that gas-filled RF multipoles exhibit increased ion transmission efficiency due to collisional ion focusing effects. This unexpected experimental finding was later supported by elementary Monte Carlo simulations. Both experiments and simulations assumed a resting background gas with typical pressures of the order of 1 Pa. However, considerable additional improvements can be achieved if laser desorbed sample ions are introduced immediately after desorption, still within the ion source, in an axisymmetric rarefied supersonic gas jet with peak pressure of the order of 100 Pa and flow velocities >300 m/s, and under weak electric fields. We describe here the design principle and report performance data of an ion source coined "MALDI-2," which incorporates elements of both rarefied aerodynamics and particle optics. Such a design allows superb suppression of metastable fragmentation due to rapid collisional cooling in <10 μs and nearly perfect injection efficiency into the attached RF ion guide, as numerous experiments have confirmed.

Production of excited H atoms at the C 1sedge of the methane molecule studied by VUV-photon–photoion and metastable-fragment–photoion coincidence experiments

Coincidences between neutral particles and photoions have been measured at the C $1s$ edge of the CH${}_{4}$ molecule. These neutral particles are either vacuum ultraviolet (VUV) photons or metastable fragments and their contributions can be separated temporally. Observed VUV-photon--photoion coincidences were mostly triggered by Lyman emission in excited H atoms. Two metastable fragment-photoion channels were observed and assigned to H${}^{*}$-H${}^{+}$ and H${}^{*}$-CH${}_{n}{}^{+}$ ($n=0--3$) coincidences, where H${}^{*}$ signifies a metastable H atom. The coincidence yields as a function of photon energy revealed all the core excitations, but were particularly enhanced just above the C $1s$ ionization threshold and also displayed the vibrational structure related to the core-ionized C $1{s}^{\ensuremath{-}1}$ state. The metastable H atoms observed in the present study were produced in highly excited Rydberg states and could be partly field ionized. The approximate kinetic energy distributions of the metastable H${}^{*}$ atoms have been derived from the H${}^{*}$-H${}^{+}$ coincidence measurements performed at the C $1s\ensuremath{\rightarrow}3p$ and $3d$ resonances as well as just above the C $1s$ threshold.