Infrared Multiphoton Dissociation Research Articles

Top-Down Deep Sequencing of Ubiquitin Using Two-Dimensional Mass Spectrometry.

Two-dimensional mass spectrometry (2DMS) allows data independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors without isolation prior to fragmentation. Developments in computer capabilities and implementations in Fourier transform ion cyclotron resonance (FTICR) MS over the past decade have allowed the technique to become a useful analytical tool for bottom-up proteomics (BUP) and, more recently, in top-down protein analysis (TDP). In this work, a new method of TDP is developed using 2D FTICR MS, called MS/2D FTICR MS or MS/2DMS. In MS/2DMS, an entire protein is initially fragmented in a hexapole collision cell, e.g., with collisionally activated dissociation (CAD). The primary fragments are then sent to the ICR cell, where 2DMS is performed with infrared multiphoton dissociation (IRMPD) or electron-capture dissociation (ECD). The resulting 2D mass spectra retain information equivalent to a set of TDP MS3 experiments on the selected protein. Up to n - 1 fragmentation steps can be added to the process, as long as an ion of interest can be unambiguously fragmented before the ICR cell, leading to an MS n/2DMS experiment whose output is a 2D mass spectrum retaining information equivalent to MS n. MS/2DMS and MS/MS/2DMS are used in this work for the structural analysis of ubiquitin (Ubi), noting several unique features which aid fragment identification. The use of CAD-MS/IRMPD-2DMS, CAD-MS/ECD-2DMS, and MS2/2DMS using, respectively, in-source dissociation (ISD), CAD, and ECD-2DMS led to 97% cleavage coverage for Ubi.

An Unprecedented Retro-Mumm Rearrangement Revealed by ESI-MS/MS, IRMPD Spectroscopy, and DFT Calculations.

Brønsted acids and protic solvents mediate acyl transfer, known as the Mumm rearrangement, from imidates to the corresponding acylamides. This represents a key step in several reactions, for example, the Ugi four-component reaction (U-4CR) and Passerini three-component reaction (P-3CR). Herein, an unprecedented break of the non-reversibility of the Mumm rearrangement is reported. A combination of electrospray tandem mass spectrometry (ESI-MS/MS), infrared multiphoton dissociation (IRMPD) ion spectroscopy and theoretical calculations demonstrates the occurrence of the retro-Mumm rearrangement of protonated isopeptides in the gas phase. In the gas phase, the extra proton acquired during ESI promotes the backward isomerisation reaction in a catalytic fashion.

Uranyl/12-crown-4 Ether Complexes and Derivatives: Structural Characterization and Isomeric Differentiation.

The following gas-phase uranyl/12-crown-4 (12C4) complexes were synthesized by electrospray ionization: [UO2(12C4)2]2+ and [UO2(12C4)2(OH)]+. Collision-induced dissociation (CID) of the dication resulted in [UO2(12C4-H)]+ (12C4-H is a 12C4 that has lost one H), which spontaneously adds water to yield [UO2(12C4-H)(H2O)]+. The latter has the same composition as complex [UO2(12C4)(OH)]+ produced by CID of [UO2(12C4)2(OH)]+ but exhibits different reactivity with water. The postulated structures as isomeric [UO2(12C4-H)(H2O)]+ and [UO2(12C4)(OH)]+ were confirmed by comparison of infrared multiphoton dissociation (IRMPD) spectra with computed spectra. The structure of [UO2(12C4-H)]+ corresponds to cleavage of a C-O bond in the 12C4 ring, with formation of a discrete U-Oeq bond and equatorial coordination by three intact ether moieties. Comparison of IRMPD and computed IR spectra furthermore enabled assignment of the structures of the other complexes. Theoretical studies of the chemical bonding features of the complexes provide an understanding of their stabilities and reactivities. The results reveal bonding and structures of the uranyl/12C4 complexes and demonstrate the synthesis and identification of two different isomers of gas-phase uranyl coordination complexes.

Identification of the fragment of the 1-methylpyrene cation by mid-IR spectroscopy

The fragment of the 1-methylpyrene cation, C17H11+, is expected to exist in two isomeric forms, 1-pyrenemethylium PyrCH2+ and the tropylium containing species PyrC7+. We measured the infrared (IR) action spectrum of cold C17H11+ tagged with Ne using a cryogenic ion trap instrument coupled to the FELIX laser. Comparison of the experimental data with density functional theory calculations allows us to identify the PyrCH2+ isomer in our experiments. The IR Multi-Photon Dissociation spectrum was also recorded following the C2H2 loss channel. Its analysis suggests combined effects of anharmonicity and isomerisation while heating the trapped ions, as shown by molecular dynamics simulations.

Frontispiece: Infrared Multiphoton Dissociation Spectroscopy with Free‐Electron Lasers: On the Road from Small Molecules to Biomolecules

Frontispiece: Infrared Multiphoton Dissociation Spectroscopy with Free‐Electron Lasers: On the Road from Small Molecules to Biomolecules

Infrared Multiphoton Dissociation Spectroscopy with Free-Electron Lasers: On the Road from Small Molecules to Biomolecules.

Infrared multiphoton dissociation (IRMPD) spectroscopy is commonly used to determine the structure of isolated, mass-selected ions in the gas phase. This method has been widely used since it became available at free-electron laser (FEL) user facilities. Thus, in this Minireview, we examine the use of IRMPD/FEL spectroscopy for investigating ions derived from small molecules, metal complexes, organometallic compounds and biorelevant ions. Furthermore, we outline new applications of IRMPD spectroscopy to study biomolecules.

A Comparative IRMPD and DFT Study of Fe3+ and UO22+ Complexation with N-Methylacetohydroxamic Acid.

Iron(III) and uranyl complexes of N-methylacetohydroxamic acid (NMAH) have been investigated by mass spectrometry, infrared multiphoton dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations. A comparison between IRMPD and theoretical IR spectra enabled one to probe the structures for some selected complexes detected in the gas phase. The results show that coordination of Fe3+ and UO22+ by hydroxamic acid is of a very similar nature. Natural bond orbital analysis suggests that bonding in uranyl complexes possesses a slightly stronger ionic character than that in iron complexes. Collision-induced dissociation (CID), IRMPD, and 18O-labeling experiments unambiguously revealed a rare example of the U═O bond activation concomitant with the elimination of a water molecule from the gaseous [UO2(NMA)(NMAH)2]+ complex. The U═O bond activation is observed only for complexes with one monodentate NMAH molecule forming a hydrogen bond toward one "yl" oxygen atom, as was found by DFT calculations. This reactivity might explain oxygen exchange observed for uranyl complexes.

Self-assembled uracil complexes containing tautomeric uracils: an IRMPD spectroscopic and computation study of the structures of gaseous uracilnCa2+ (n = 4, 5, or 6) complexes.

The structures of doubly-charged uracil (U) complexes with Ca2+, UnCa2+ (n = 4, 5, 6), were studied by infrared multiple photon dissociation (IRMPD) spectroscopy and computational methods. The ions were produced by electrospray ionization (ESI) and were isolated in the gas phase in a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR-MS). The recorded IRMPD spectra in both the fingerprint and the C-H/N-H/O-H stretching regions, combined with computed vibrational spectra, reveal that the structures present in the greatest abundance consist of both canonical uracil as well as the lactam (or colloquially "enol") tautomer of uracil. U4Ca2+ consists of two hydrogen-bonded dimers of uracil, one canonical and one tautomer, with each uracil interacting with Ca2+ through a carbonyl oxygen. The structures most consistent with the vibrational spectrum of U6Ca2+ consist of two hydrogen-bonded uracil trimers, each composed of two canonical and one enolic uracil, with each uracil also interacting with Ca2+ through carbonyl oxygen. U5Ca2+ consists of one of the aforementioned trimers and dimers, each containing one enol tautomerized uracil. The computed structures whose vibrational spectra best agree with the experimental vibrational spectra are also the lowest-energy structures for all three complexes. This study clearly shows that some uracils adopt the normally very high energy enol tautomer in the lowest energy gas phase complexes of uracil with a doubly-charged ion like Ca2+.

Determination of soyasaponins in Fagioli di Sarconi beans (Phaseolus vulgaris L.) by LC-ESI-FTICR-MS and evaluation of their hypoglycemic activity.

Soyasaponins are oleanene-type triterpenoid saponins, naturally occurring in many edible plants that have attracted a great deal of attention for their role in preventing chronic diseases. The aim of this study was to establish the distribution and the content of soyasaponins in 21 ecotypes of Fagioli di Sarconi beans (Phaseolus vulgaris, Leguminosae). High-performance reversed-phase liquid chromatography (RPLC) with positive electrospray ionization (ESI(+)) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) in conjunction with infrared multiphoton dissociation (IRMPD) was applied for the unambiguous identification of soyasaponins Ba (m/z 959.5213, [C48H79O19]+), Bb (m/z 943.5273, [C48H79O18]+), Bd (m/z 957.5122, [C48H77O19]+), and Be (m/z 941.5166, [C48H77O18]+), which are the only commercially available reference standards. In addition, the several diagnostic product ions generated by IRMPD in the ICR-MS cell allowed us the putative identification of soyasaponins Bb' (m/z 797.4680, [C42H69O14]+), αg (m/z 1085.5544, [C54H85O22]+), βg (m/z 1069.5600, [C54H85O21]+), and γg (m/z 923.5009, [C48H75O17]+), establishing thus their membership in the soyasaponin group. Quantitative and semiquantitative analysis of identified soyasaponins were also performed by RPLC-ESI(+) FTICR-MS; the total concentration levels were found ranging from 83.6 ± 9.3 to 767 ± 37mg/kg. In vitro hypoglycemic outcomes of four soyasaponin standards were evaluated; significant inhibitory activities were obtained with IC50 values ranging from 1.5 ± 0.1 to 2.3 ± 0.2μg/mL and 12.0 ± 1.1 to 29.4 ± 1.4μg/mL for α-glucosidase and α-amylase, respectively. This study represents the first detailed investigation on the antidiabetic activity of bioactive constituents found in Fagioli di Sarconi beans. Graphical abstract The first detailed RPLC-ESI(+) FTICR-MS investigation of the qualitative and semiquantitative profile of soyasaponins, occurring in 21 ecotypes of Fagioli di Sarconi beans (P. vulgaris L.).

Fingerprints of Both Watson-Crick and Hoogsteen Isomers of the Isolated (Cytosine-Guanine)H+ Pair.

Gas phase protonated guanine-cytosine (CGH+) pair was generated using an electrospray ionization source from solutions at two different pH (5.8 and 3.2). Consistent evidence from MS/MS fragmentation patterns and differential ion mobility spectra (DIMS) point toward the presence of two isomers of theCGH+ pair, whose relative populations depend strongly on the pH of the solution. Gas phase infrared multiphoton dissociation (IRMPD) spectroscopy in the 900-1900cm-1 spectral range further confirms that the Watson-Crick isomer is preferentially produced (91%) at pH = 5.8, while the Hoogsteen isomer predominates (66%) at pH = 3.2). These fingerprint signatures are expected to be useful for the development of new analytical methodologies and to trigger isomer selective photochemical studies of protonated DNA base pairs.

Bottom-Up Two-Dimensional Electron-Capture Dissociation Mass Spectrometry of Calmodulin.

Two-dimensional mass spectrometry (2D MS) is a tandem mass spectrometry technique that allows data-independent fragmentation of all precursors in a mixture without previous isolation, through modulation of the ion cyclotron frequency in the ICR-cell prior to fragmentation. Its power as an analytical technique has been proven particularly for proteomics. Recently, a comparison study between 1D and 2D MS has been performed using infrared multiphoton dissociation (IRMPD) on calmodulin (CaM), highlighting the capabilities of the technique in both top-down (TDP) and bottom-up proteomics (BUP). The goal of this work is to expand this study on CaM using electron-capture dissociation (ECD) 2D MS as a single complementary BUP experiment in order to enhance the cleavage coverage of the protein under analysis. By adding the results of the BUP 2D ECD MS to the 2D IRMPD MS analysis of CaM, the total cleavage coverage increased from ~40% to ~68%. Graphical abstract ᅟ.

Spectroscopic Discrimination of Diastereomeric Complexes Involving an Axially Chiral Receptor.

The infrared multiphoton dissociation (IRMPD) spectra of electrospray ionization (ESI)-formed proton-bound complexes between the axially chiral multifunctional macrocycle MaR and d- and l-phenylalanine (PD and PL ) or d- and l-3,4-dihydroxyphenylalanine (DD and DL ) are recorded in the ν˜ =2800-3700 cm-1 region. Whereas the diastereomeric [MaR ⋅H⋅PD ]+ and [MaR ⋅H⋅PL ]+ complexes do not show any significant spectral differences, the spectrum of [MaR ⋅H⋅DD ]+ clearly diverges from that of its [MaR ⋅H⋅DL ]+ diastereomer. A comparison of the experimental spectra with the B3LYP/6-31G**-calculated harmonic vibrational frequencies of a number of stable structures indicates the formation, in the ESI source, of very similar spectroscopically active structures, with the protonated amino acid placed outside the flat cavity of the macrocycle and hydrogen-bonded at its O2 center. Different spectral signatures of the [MaR ⋅H⋅DD ]+ and [MaR ⋅H⋅DL ]+ complexes are attributed to the coexistence of several stable rotamers in the ESI source.

Targeted Annotation of S-Sulfonylated Peptides by Selective Infrared Multiphoton Dissociation Mass Spectrometry.

Protein S-sulfinylation (R-SO2-) and S-sulfonylation (R-SO3-) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 μm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site.

Application of higher energy collisional dissociation (HCD) to the fragmentation of new DOTA-based labels and N-termini DOTA-labeled peptides.

1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives are applied in quantitative proteomics owing to their ability to react with different functional groups, to harbor lanthanoides and hence their compatibility with molecular and elemental mass spectrometry. The new DOTA derivatives, namely Ln-MeCAT-Click and Ln-DOTA-Dimedone, allow efficient thiol labeling and targeting sulfenation as an important post-translational modification, respectively. Quantitative applications require the investigation of fragmentation behavior of these reagents. Therefore, the fragmentation behavior of Ln-MeCAT-Click and Ln-DOTA-Dimedone was studied using collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and higher-energy collision dissociation (HCD) using different energy levels, and the efficiency of reporter ion production was estimated. The efficiency of characteristic fragment formation was in the order IRMPD>HCD (normal energy level)>CID. On the other hand, the application of HCD at high energy levels (HCD@HE; NCE>250%) resulted in a significant increase in reporter ion production (33-54%). This new strategy was successfully applied to generate label-specific reporter ions for DOTA amino labeling at the N-termini and in a quantitative fashion for the estimation of amino:thiol ratio in peptides. Copyright © 2017 John Wiley & Sons, Ltd.

Fragmentation behavior of DOTA complexes under different activation conditions.

We have investigated the fragmentation behavior of a number of DOTA-metal complexes under collision-induced dissociation, infrared-multiphoton dissociation and higher-energy collisional dissociation activation conditions. Both, positive and negative ion mode electrospray ionization was applied. The results show that characteristic fragmentations were obtained for the metal-complexes under the investigated conditions. All elemental compositions of fragment ions have been unambiguously identified by high resolution-accurate mass measurements. Certain trends, for instance eliminations of carbon dioxide, alkyl and amine residues, were observed that coincide with the size of the metal and its location within the periodic table. Additionally, lanthanide, aluminium and indium species with even oxidation state or containing a radical have been detected. To further investigate the observed water capture during activation, deuterium labeling experiments have also been carried out. Copyright © 2017 John Wiley & Sons, Ltd.

Implementation of Activated Ion Electron Transfer Dissociation on a Quadrupole-Orbitrap-Linear Ion Trap Hybrid Mass Spectrometer.

Using concurrent IR photoactivation during electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), significantly increases dissociation efficiency resulting in improved overall performance. Here we describe the first implementation of AI-ETD on a quadrupole-Orbitrap-quadrupole linear ion trap (QLT) hybrid MS system (Orbitrap Fusion Lumos) and demonstrate the substantial benefits it offers for peptide characterization. First, we show that AI-ETD can be implemented in a straightforward manner by fastening the laser and guiding optics to the instrument chassis itself, making alignment with the trapping volume of the QLT simple and robust. We then characterize the performance of AI-ETD using standard peptides in addition to a complex mixtures of tryptic peptides using LC-MS/MS, showing not only that AI-ETD can nearly double the identifications achieved with ETD alone but also that it outperforms the other available supplemental activation methods (ETcaD and EThcD). Finally, we introduce a new activation scheme called AI-ETD+ that combines AI-ETD in the high pressure cell of the QLT with a short infrared multiphoton dissociation (IRMPD) activation in the low-pressure cell. This reaction scheme introduces no addition time to the scan duty cycle but generates MS/MS spectra rich in b/y-type and c/z•-type product ions. The extensive generation of fragment ions in AI-ETD+ substantially increases peptide sequence coverage while also improving peptide identifications over all other ETD methods, making it a valuable new tool for hybrid fragmentation approaches.

Infrared Spectra of Hexa-peri-hexabenzocoronene Cations: HBC+ and HBC2+

Abstract We present the first infrared (IR) gas-phase spectrum of a large and astronomically relevant polycyclic aromatic hydrocarbon (PAH) cation (C42H18 +, HBC+) and its dication (C42H18 , HBC2+). The spectra are recorded via infrared multiphoton dissociation (IRMPD) spectroscopy of ions stored in a quadrupole ion trap, using the intense IR radiation of a free electron laser in the 530–1800 cm−1 (5.6–18.9 μm) range. HBC+ shows main intense absorption peaks at 762 (13.12), 1060 (9.43), 1192 (8.39), 1280 (7.81), 1379 (7.25), and 1530 (6.54) cm−1 (μm), in good agreement with density functional theory calculations after scaling to take the anharmonicities effect into account. HBC2+ has its main absorption peaks at 660 (15.15), 766 (13.05), 1054 (9.49), 1176 (8.50), 1290 (7.75), 1370 (7.30) and 1530 (6.54) cm−1 (μm). Given the similarity in the cationic and dicationic spectra, we have not identified an obvious diagnostic signature to the presence of multiply charged PAHs in space. While experimental issues associated with the IRMPD technique preclude a detailed comparison with interstellar spectra, we do note that the strong bands of HBC+ and HBC2+ at ∼6.5, 7.7, 8.4, and 13.1 μm coincide with prominent aromatic infrared bands (AIBs). HBC has only trio CH groups, and the out-of-plane CH bending mode of both HBC cations is measured at 13.1 μm, squarely in the range predicted by theory and as previously found in studies of small (substituted) PAHs. This study therefore supports the use of AIBs observed in the 11–14 μm range as a diagnostic tool for the edge topology of large PAHs in space.

Ergothioneine and related histidine derivatives in the gas phase: tautomer structures determined by IRMPD spectroscopy and theory.

l-Ergothioneine (ET) is a sulfur-containing derivative of the amino acid histidine that offers unique antioxidant properties. The enzyme independent redox-chemistry of ET relies on the availability of the thiol tautomer to allow oxidative formation of disulfide bridges, i.e., the tautomeric equilibrium. To study the intrinsic properties of ET the tautomeric equilibrium is studied in the gas-phase by infrared multiphoton dissociation (IRMPD) spectroscopy. The IR ion spectra of isolated molecular ions of ET and of the biosynthetic precursors of ET, i.e., hercynine and Nε-methyl-hercynine are acquired. The analyte structures are independently investigated by density functional theory (DFT) and computed linear IR-spectra of tautomer ion structures are compared with the gas-phase spectra for identification. For the molecular ion of ET the simulated IR spectra of thione and thiol structures match the recorded IRMPD spectrum and that prevents an individual structure assignment. On the other hand, theory suggests that ET adopts a thione tautomer in MeOH solution which could be carried over from the condensed phase to gas phase and could be kinetically trapped after effective electrospray phase transfer and desolvation. Such a non-thermal behavior is also found for the molecular ions of protonated hercynine and Nε-methyl-hercynine. Contrary to that, the sodium complex ions of ET, hercynine and Nε-methyl-hercynine adopt the respective ground structures predicted by theory, which are reliably identified spectroscopically. For ET the thione tautomer is by far the most stable isomer in the sodium complex molecular ion.

IRMPD Spectroscopy of Metalated Flavins: Structure and Bonding of Lumiflavin Complexes with Alkali and Coinage Metal Ions.

Flavins are a fundamental class of biomolecules, whose photochemical properties strongly depend on their environment and their redox and metalation state. Infrared multiphoton dissociation (IRMPD) spectra of mass-selected isolated metal-lumiflavin ionic complexes (M+LF) are analyzed in the fingerprint range (800-1830 cm-1) to determine the bonding of lumiflavin with alkali (M = Li, Na, K, Cs) and coinage (M = Cu, Ag) metal ions. The complexes are generated in an electrospray ionization source coupled to an ion cyclotron resonance mass spectrometer and the IR free electron laser FELIX. Vibrational and isomer assignments of the IRMPD spectra are accomplished by comparison to quantum chemical calculations at the B3LYP/cc-pVDZ level, yielding structure, binding energy, bonding mechanism, and spectral properties of the complexes. The most stable binding sites identified in the experiments involve metal bonding to the oxygen atoms of the two available CO groups of LF. Hence, CO stretching frequencies are a sensitive indicator of both the metal binding site and the metal bond strength. More than one isomer is observed for M = Li, Na, and K, and the preferred CO binding site changes with the size of the alkali ion. For Cs+LF, only one isomer is identified, although the energies of the two most stable structures differ by less than 7 kJ/mol. While the M+-LF bonds for alkali ions are mainly based on electrostatic forces, substantial covalent contributions lead to stronger bonds for the coinage metal ions. Comparison between lumiflavin and lumichrome reveals substantial differences in the metal binding motifs and interactions due to the different flavin structures.

Fragmentation study of major spirosolane‐type glycoalkaloids by collision‐induced dissociation linear ion trap and infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry

Glycoalkaloids play a key role in the plant protection system against phytopathogens including fungi, viruses, bacteria, insects and worms. They can be toxic to humans if consumed in high concentrations causing gastrointestinal disturbances. The structural characterization of the major spirosolane glycoalkaloids, solasonine, solamargine, α-tomatine and dehydrotomatine, were investigated by positive electrospray ionization (ESI) coupled with a hybrid linear ion trap (LIT) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Tandem mass spectrometric analysis of spirosolane glycoalkaloids was performed by both collision-induced dissociation (CID) within the LIT and infrared multiphoton dissociation (IRMPD) in conjunction with the FTICR cell. Several common product ions were observed, generated by losses of the sugar moiety or aglycone fragmentation in the B- or E-ring, that can provide information on the accurate mass of aglycone and the primary sequence and branching of the oligosaccharide chains. Thanks to the multistage CID it was possible to understand the fragmentation pathways and thanks to the high resolution of IRMPD-FTICR the elemental compositions of product ions were obtained. Because the investigated tandem mass spectra data were acquired with high mass accuracy, unambiguous interpretation and determination of the chemical compositions for the majority of detected fragment ions were feasible. From these data, generalized fragmentation pathways were proposed, providing guidance for the characterization of unknown glycoalkaloids in plants. Copyright © John Wiley & Sons, Ltd.