Infrared Multiphoton Dissociation Research Articles

Structural Elucidation of Ubiquitin via Gas-Phase Ion/Ion Cross-Linking Reactions Using Sodium-Cationized Reagents Coupled with Infrared Multiphoton Dissociation.

Accurate structural determination of proteins is critical to understanding their biological functions and the impact of structural disruption on disease progression. Gas-phase cross-linking mass spectrometry (XL-MS) via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions has previously been developed to obtain low-resolution structural information on proteins. This method significantly shortens experimental time relative to conventional solution-phase XL-MS but has several technical limitations: (1) the singly deprotonated N-hydroxysulfosuccinimide (sulfo-NHS)-based cross-linker anions are restricted to attachment at neutral amine groups of basic amino acid residues and (2) analyzing terminal cross-linked fragment ions is insufficient to unambiguously localize sites of linker attachment. Herein, we demonstrate enhanced structural information for alcohol-denatured A-state ubiquitin obtained from an alternative gas-phase XL-MS approach. Briefly, singly sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions enable covalent cross-linking at both ammonium and amine groups. Additionally, covalently modified internal fragment ions, along with terminal b-/y-type counterparts, improve the determination of linker attachment sites. Molecular dynamics simulations validate experimentally obtained gas-phase conformations of denatured ubiquitin. This method has identified four cross-linking sites across 8+ ubiquitin, including two new sites in the N-terminal region of the protein that were originally inaccessible in prior gas-phase XL approaches. The two N-terminal cross-linking sites suggest that the N-terminal half of ubiquitin is more compact in gas-phase conformations. By comparison, the two C-terminal linker sites indicate the signature transformation of this region of the protein from a native to a denatured conformation. Overall, the results suggest that the solution-phase secondary structures of the A-state ubiquitin are conserved in the gas phase. This method also provides sufficient sensitivity to differentiate between two gas-phase conformers of the same charge state with subtle structural variations.

Structural connectivity of 18‐Crown‐6/H+/L‐tryptophan noncovalent complexes in gas phase and in solution: Delineating host–guest–solvent interactions in solution from gas phase structures

AbstractWe investigate the structural correlation of noncovalent crown ether/H+/L‐tryptophan (CR/TrpH+) host–guest complexes in the solution phase with those in the gas phase generated through electrospray ionization/mass spectrometry (ESI/MS) techniques. We perform quantum chemical calculations to determine their structures, relative Gibbs free energies, and infrared spectra. We compare the calculated infrared (IR) spectra with the IR multiphoton dissociation (IRMPD) spectra observed for the 18‐Crown‐6/TrpH+ complex by Polfer and co‐workers [J. Phys. Chem. A 2013, 117, 1181–1188] for assigning the IR bands. We observe that the carboxyl group remains “naked,” lacking hydrogen bonding with the CR unit in the gas phase, and that this most stable conformer originates from the corresponding lowest Gibbs free energy structure in solution. Based on these findings, we propose that gas phase host–guest complexes directly correlate with those in solution, reinforcing the possibility of obtaining invaluable information about host–guest–solvent interactions in solution from the structure of the host–guest pair in the gas phase.

Relative Quantification of Lipid Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions and Infrared Multiphoton Dissociation.

Accurate structural identification of lipids in imaging mass spectrometry is critical to properly contextualizing spatial distributions with tissue biochemistry. Gas-phase charge inversion ion/ion reactions alter the ion type prior to dissociation to allow for more structurally informative fragmentation and improve lipid identification at the isomeric level. In this work, infrared multiphoton dissociation (IRMPD) was interfaced with a commercial hybrid Qh-FT-ICR mass spectrometer to enable the rapid fragmentation of gas-phase charge inversion ion/ion reaction products at every pixel in imaging mass spectrometry experiments. An ion/ion reaction between phosphatidylcholine (PC) monocations generated from rat brain tissue via matrix-assisted laser desorption/ionization (MALDI) and 1,4-phenylenediproprionic acid reagent dianions generated via electrospray ionization (ESI) followed by IRMPD of the resulting product ion complex produces selective fatty acyl chain cleavages indicative of fatty acyl carbon compositions in the lipid. Ion/ion reaction images using this workflow allow for mapping of the relative spatial distribution of multiple PC isomers under a single sum composition lipid identification. Lipid isomers display significantly different relative spatial distributions within rat brain tissue, highlighting the importance of resolving isomers in imaging mass spectrometry experiments.

Structural Characterization of the Metalized Radical Cations of Adenosine ([Ade+Li-H]•+ and [Ade+Na-H]•+) by Infrared Multiphoton Dissociation Spectroscopy and Theoretical Studies.

Nucleoside radicals are key intermediates in the process of DNA damage, and alkali metal ions are a common group of ions in living organisms. However, so far, there has been a significant lack of research on the structural effects of alkali metal ions on nucleoside free radicals. In this study, we report a new method for generating metalized nucleoside radical cations in the gas phase. The radical cations [Ade+M-H]•+ (M = Li, Na) are generated by the 280 nm ultraviolet photodissociation (UVPD) of the precursor ions of lithiated and sodiated ions of 2-iodoadenine in a Fourier transform ion cyclotron resonance (FT ICR) cell. Further infrared multiphoton dissociation (IRMPD) spectra of both radical cations were recorded in the region of 2750-3750 cm-1. By combining these results with theoretical calculations, the most stable isomers of both radicals can be identified, which share the common characteristics of triple coordination patterns of the metal ions. For both radical species, the lowest-energy isomers undergo hydrogen transfer. Although the sugar ring in the most stable isomer of [Ade+Li-H]•+ is in a (South, syn) conformation similar to that of [Ado+Na]+, [Ade+Na-H]•+ is distinguished by the unexpected opening of the sugar ring. Their theoretical spectra are in good agreement with experimental spectra. However, due to the flexibility of the structures and the complexity of their potential energy surfaces, the hydrogen transfer pathways still need to be further studied. Considering that the free radicals formed directly after C-I cleavage have some similar spectral characteristics, the existence of these corresponding isomers cannot be ruled out. The findings imply that the structures of nucleoside radicals may be significantly influenced by the attached alkali metal ions. More detailed experiments and theoretical calculations are still crucial.

Understanding the fragmentation of glucose in mass spectrometry.

The fragmentation mechanism of D-glucose was investigated in detail by two different fragmentation techniques, namely, collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) using all six 13 C-labeled isotopomers and 2 H-labeled isotopomers. For both CID and IRMPD energy-resolved measurements were carried out. Individual fragmentation pathways were studied at MS2 and MS3 levels. Additionally, we have developed an HPLC-tandem MS method to separate the anomers of D-glucose using a HILIC column and investigated their fragmentation patterns individually. We propose a complete fragmentation landscape of D-glucose, demonstrating that a rather simple multifunctional molecule displays extreme complexity in gas phase dissociation, following multiple parallel fragmentation routes yielding a total of 23 distinct fragment ions. The results allowed a detailed formulation of the complex fragmentation mechanism of D-glucose. The results have immediate consequences for the full structure analysis of complex carbohydrates.

Characterization of an Omnitrap-Orbitrap Platform Equipped with Infrared Multiphoton Dissociation, Ultraviolet Photodissociation, and Electron Capture Dissociation for the Analysis of Peptides and Proteins.

We describe an instrument configuration based on the Orbitrap Exploris 480 mass spectrometer that has been coupled to an Omnitrap platform. The Omnitrap possesses three distinct ion-activation regions that can be used to perform resonant-based collision-induced dissociation, several forms of electron-associated fragmentation, and ultraviolet photodissociation. Each section can also be combined with infrared multiphoton dissociation. In this work, we demonstrate all these modes of operation in a range of peptides and proteins. The results show that this instrument configuration produces similar data to previous implementations of each activation technique and at similar efficiency levels. We demonstrate that this unique instrument configuration is extremely versatile for the investigation of polypeptides.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

Distinguishing gas phase lactose and lactulose complexed with sodiated L-arginine by IRMPD spectroscopy and DFT calculations.

We present the origin of the observed differentiation of lactose and lactulose achieved by complexation with sodiated L-arginine (ArgNa+). We find that the infrared multiphoton dissociation (IRMPD) bands in 3600-3650 and >3650 cm-1 regimes for gas phase lactose and lactulose, respectively, vanish when forming host-guest complexes with ArgNa+. We interpret these differences in the IRMPD spectra by scrutinizing the interactions between the functional groups (guanidium, -CO2-Na+) in ArgNa+ and -OHs in lactose/lactulose. Our calculated structures and infrared spectra of lactose/ArgNa+ and lactulose/ArgNa+ host-guest pairs indicate that the functional groups interact with the low- and high-frequency -OH stretch modes of lactose and lactulose, respectively, in the 3600-3720 cm-1 window.

Multimodal Tandem Mass Spectrometry Techniques for the Analysis of Phosphopeptides.

Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging.

Differentiation of Dihydroxylated Vitamin D3 Isomers Using Tandem Mass Spectrometry.

Vitamin D compounds are a group of secosteroids derived from cholesterol that are vital for maintaining bone health in humans. Recent studies have shown extraskeletal effects of vitamin D, involving vitamin D metabolites such as the dihydroxylated vitamin D3 compounds 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Differentiation and characterization of these isomers by mass spectrometry can be challenging due to the zero-mass difference and minor structural differences between them. The isomers usually require separation by liquid chromatography (LC) prior to mass spectrometry, which adds extra complexity to the analysis. Herein, we investigated and revisited the use of fragmentation methods such as collisional induced dissociation (CID), infrared multiphoton dissociation (IRMPD), electron induced dissociation (EID), and ultraviolet photodissociation (UVPD), available on a 12T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) to generate characteristic fragments for the dihydroxylated vitamin D3 isomers that can be used to distinguish between them. Isomer-specific fragments were observed for the 1,25-dihydroxyvitamin D3, which were clearly absent in the 24,25-dihydroxyvitamin D3 MS/MS spectra using all fragmentation methods mentioned above. The fragments generated due to cleavage of the C-6/C-7 bond in the 1,25-dihydroxyvitamin D3 compound demonstrate that the fragile OH groups were retained during fragmentation, thus enabling differentiation between the two dihydroxylated vitamin D3 isomers without the need for prior chromatographic separation or derivatization.

Efficient two-stage laser enrichment up to 99% of carbon-13 isotope by IR multiphoton dissociation of Freon molecules

A new method for two-stage laser enrichment of carbon-13 (13C) by isotope-selective IR multiphoton dissociation of Freon molecules is considered, which makes it possible to achieve a concentration of 13C up to 99 % or higher with high productivity. At the first stage, as a result of the selective dissociation of CF2HCl (Freon-22) molecules, it is expected to obtain a C2F4 dissociation product enriched in 13C up to 30 % – 50 %. At the second stage, CF2Br2 (Freon-12B2) is proposed to be used as a working substance, which is synthesised from enriched C2F4, and the enrichment process is carried out by selective deep ‘burning-out’ of the 12C-containing component. Experiments on multiphoton dissociation of Freon-12B2 with an initial 13CF2Br2 concentration of 30 % in a mixture with oxygen by pulsed CO2 laser radiation are performed. It is shown that reaching a 13CF2Br2 concentration of no lower than 99 % is possible at high values of the elementary separation act parameters: 15 % – 18 % dissociation yield and 40 – 75 selectivity for 12CF2Br2 molecules.

FTIR and Raman Spectroscopy Study of Soot Deposits Produced in the Infrared Multiphoton Dissociation of Vinyl Bromide

Even at low concentrations, poly-aromatic hydrocarbons found in soot have substantial health implications. Soot deposits have been reported and studied using FTIR and Raman spectroscopy. Using a CO2 photolysis laser, the samples were obtained via infrared multiphoton dissociation (IRMPD) of vinyl bromide (VBr, C2H3Br) molecules. The solid deposit formed in the IRMPD of VBr when a relatively high fluence of the order of 204 J.cm−2 was analyzed by FTIR, and it was discovered that the majority of its composition is aromatic and aliphatic hydrocarbons. Significant fullerene-type carbonaceous soot particles are also found, which could correspond to C60 and C70 or other carbonaceous agglomerates of a higher order; however, the disappearance of this fullerene on Raman spectra cast doubt on this explanation. Our samples’ Raman spectroscopy has been compared to Tamor and Vassell’s research, which may indicate that they have a lesser degree of hardness and density than these authors’ results, indicating a larger hydrogen content in our samples. The optical gap has been calculated, yielding a very limited range of values ranging only between 1.0 and 1.2 eV, resulting in a crystalline size of 0.58 to 1.12 nm.

Hemp Chemotype Definition by Cannabinoids Characterization Using LC-ESI(+)-LTQ-FTICR MS and Infrared Multiphoton Dissociation

The development and application of advanced analytical methods for a comprehensive analysis of Cannabis sativa L. extracts plays a pivotal role in order to have a reliable evaluation of their chemotype definition to guarantee the efficacy and safety in pharmaceutical use. This paper deals with the qualitative and quantitative determination of cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), and cannabigerol (CBG) based on a liquid chromategraphy-mass spectrometry (LC-MS) method using electrospray ionization in positive mode (ESI+), coupled with a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). For the first time, structural information of phytocannabinoids is available upon precursor ions’ isolation within the FTICR trapping cell and subsequent fragmentation induced by infrared multiphoton dissociation (IRMPD). Such fragmentation and accurate mass measurement of product ions, alongside collision-induced dissociation (CID) within LTQ, was advantageous to propose a reliable fragmentation pattern for each compound. Then, the proposed LC-ESI(+)-LTQ-FTICR MS method was successfully applied to the hemp chemotype definition of three registered Italian accessions of hemp C. sativa plants (Carmagnola C.S., Carmagnola, and Eletta Campana), thus resulting in the Eletta Campana accession being the best one for cannabis product manufacturing.

IRMPD Spectroscopy of Homo- and Heterochiral Asparagine Proton-Bound Dimers in the Gas Phase.

We investigate gas-phase structures of homo- and heterochiral asparagine proton-bound dimers with infrared multiphoton dissociation (IRMPD) spectroscopy and quantum-chemical calculations. Their IRMPD spectra are recorded at room temperature in the range of 500–1875 and 3000–3600 cm–1. Both varieties of asparagine dimers are found to be charge-solvated based on their IRMPD spectra. The location of the principal intramolecular H-bond is discussed in light of harmonic frequency analyses using the B3LYP functional with GD3BJ empirical dispersion. Contrary to theoretical analyses, the two spectra are very similar.

Combining Ultraviolet Photodissociation and Two-Dimensional Mass Spectrometry: A Contemporary Approach for Characterizing Singly Charged Agrochemicals.

Ultraviolet photodissociation (UVPD) has been shown to produce extensive structurally informative data for a variety of chemically diverse compounds. Herein, we demonstrate the performance of the 193 nm UVPD fragmentation technique on structural/moiety characterization of 14 singly charged agrochemicals. Two-dimensional mass spectrometry (2DMS) using infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID) have previously been applied to a select range of singly charged pesticides. The ≥80% moiety coverage achieved for the majority of the species by the UVPD and 2D-UVPD methods was on par with and, in some cases, superior to the data obtained by other fragmentation techniques in previous studies, demonstrating that UVPD is viable for these types of species. A three-dimensional (3D) peak picking method was implemented to extract the data from the 2DMS spectrum, overcoming the limitations of the line extraction method used in previous studies, successfully separating precursor specific fragments with milli-Dalton accuracy. Whole spectrum internal calibration combined with 3D peak picking obtained sub-part-per-million (ppm) to part-per-billion (ppb) mass accuracies across the entire 2DMS spectrum.

Fragmentation behavior of EDTA complexes under different activation conditions.

Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid and complexation agent that is able to bind a large variety of metals. The formation of highly stable metal-EDTA complexes is generally very quick. This has led to the use of EDTA in a variety of applications, including food, medical, and household applications. In the current study, we have investigated the fragmentation behavior of EDTA and various metal complexes under collision-induced dissociation (CID), infrared-multiphoton dissociation (IRMPD), and higher-energy collisional dissociation (HCD) activation conditions. Both, positive and negative mode electrospray ionization (ESI) were applied. The metals used to complex with EDTA ranged from alkaline earth metals, such as sodium and cesium, via calcium, nickel, zinc, aluminum, copper, iron, and indium to yttrium and several lanthanides. Furthermore, the protonated and deprotonated species of EDTA, as well as disodium and trisodium species, have been subjected to fragmentation. The results show that characteristic fragmentations were obtained for EDTA and the metal complexes under the investigated conditions. The use of an ion cyclotron resonance (ICR) and an Orbitrap mass spectrometer, as high resolution-accurate mass instruments, allowed the assignment of elemental compositions undoubtedly for the vast majority of fragments. Certain trends were observed that trend correlated with the size of the metal and the location within the periodic table.

Versatile Double-Beam Confocal Laser System Combined with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer for Photodissociation Mass Spectrometry and Spectroscopy.

Both infrared multiphoton dissociation (IRMPD) and ultraviolet photodissociation (UVPD) play important roles in tandem mass spectrometry and the action spectroscopy of organic and biological molecules. A flexible combination of the two methods may provide researchers with more versatile and powerful ion activation/dissociation choices for structural characterization and spectroscopic studies. Here, we report the integration of two tunable lasers with a Fourier transform ion cyclotron resonance mass spectrometer in a confocal mode, which offers multiple capabilities for photon activation/dissociation experiments. The two overlapped beams can be introduced into the cell individually, sequentially, or simultaneously, providing highly flexible and diverse activation schemes. The setup can also measure the UVPD or IRMPD action spectra of fragment ions generated by previous photon dissociation processes. In addition, the multistage tandem-in-time mass spectrometry performance up to MS4, including three different activation methods in a single cell, has also been demonstrated.

Comprehensive analysis of multiple asphaltene fractions combining statistical analyses and novel visualization tools

Marine heavy fuel oils (HFOs), derived from and often blended with hydrotreated residual cuts, typically possess high boiling point, viscosity, and molecular complexity, and so are inherently challenging to analyze at the molecular level. Their high asphaltene content is associated with undesirable phenomena including flocculation, deposition, and black paint formation in marine engines. Asphaltene fractions of eight HFOs were selected for analysis by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) due to their differing behaviour, including responsiveness to additive chemistries designed to stabilise against asphaltene handling issues. A selected mass region was isolated and fragmented using infra-red multiphoton dissociation (IRMPD), and the relative heteroatom content in asphaltene cores and smaller aromatic moieties, and degree of alkylation and aromaticity, of the fragments generated for each sample compared. A more extensive elucidation of the molecular-level differences between the n-alkane insoluble asphaltene fractions is afforded, with key components underlying variation in bulk behaviour identified through statistical approaches. The approach presented allows additive packages to be adjusted to address asphaltene handling issues presented by more challenging samples, and simpler and more ubiquitous formulations may eventually be developed.

Enhancing Top-Down Analysis Using Chromophore-Assisted Infrared Multiphoton Dissociation from (Phospho)peptides to Protein Assemblies.

Infrared multiphoton dissociation (IRMPD) has been used in mass spectrometry to fragment peptides and proteins, providing fragments mostly similar to collisional activation. Using the 10.6 μm wavelength of a CO2 laser, IRMPD suffers from the relative low absorption cross-section of peptides and small proteins. Focusing on top-down analysis, we investigate different means to tackle this issue. We first reassess efficient sorting of phosphopeptides from nonphosphopeptides based on IR-absorption cross-sectional enhancement by phosphate moieties. We subsequently demonstrate that a myo-inositol hexakisphosphate (IP6) noncovalent adduct can substantially enhance IRMPD for nonphosphopeptides and that this strategy can be extended to proteins. As a natural next step, we show that native phospho-proteoforms of proteins display a distinct and enhanced fragmentation, compared to their unmodified counterparts, facilitating phospho-group site localization. We then evaluate the impact of size on the IRMPD of proteins and their complexes. When applied to protein complexes ranging from a 365 kDa CRISPR–Cas Csy ribonucleoprotein hetero-decamer, a 800 kDa GroEL homo-tetradecamer in its apo-form or loaded with its ATP cofactor, to a 1 MDa capsid-like homo-hexacontamer, we conclude that while phosphate moieties present in crRNA and ATP molecules enhance IRMPD, an increase in the IR cross-section with the size of the protein assembly also favorably accrues dissociation yields. Overall, our work showcases the versatility of IRMPD in the top-down analysis of peptides, phosphopeptides, proteins, phosphoproteins, ribonucleoprotein assemblies, and large protein complexes.