Tandem-trapped ion mobility spectrometry/mass spectrometry coupled with ultraviolet photodissociation.

RATIONALE: Tandem-ion mobility spectrometry/mass spectrometry methods have recently gained traction for the structural characterization of proteins and protein complexes. However, ion activation techniques currently coupled with tandem-ion mobility spectrometry/mass spectrometry methods are limited in their ability to characterize structures of proteins and protein complexes. METHODS: Here, we describe the coupling of the separation capabilities of tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS) with the dissociation capabilities of ultraviolet photodissociation (UVPD) for protein structure analysis. RESULTS: We establish the feasibility of dissociating intact proteins by UV irradiation at 213 nm between the two TIMS devices in tTIMS/MS and at pressure conditions compatible with ion mobility spectrometry (2-3 mbar). We validate that the fragments produced by UVPD under these conditions result from a radical-based mechanism in accordance with prior literature on UVPD. The data suggest stabilization of fragment ions produced from UVPD by collisional cooling due to the elevated pressures used here ("UVnoD2"), which otherwise do not survive to detection. The data account for a sequence coverage for the protein ubiquitin comparable to recent reports, demonstrating the analytical utility of our instrument in mobility-separating fragment ions produced from UVPD. CONCLUSIONS: The data demonstrate that UVPD carried out at elevated pressures of 2-3 mbar yields extensive fragment ions rich in information about the protein and that their exhaustive analysis requires IMS separation post-UVPD. Therefore, because UVPD and tTIMS/MS each have been shown to be valuable techniques on their own merit in proteomics, our contribution here underscores the potential of combining tTIMS/MS with UVPD for structural proteomics.

Mechanistic insights into intramolecular phosphate group transfer during collision induced dissociation of phosphopeptides.

We report on the rearrangement chemistry of model phosphorylated peptides during collision-induced dissociation (CID), where intramolecular phosphate group transfers are observed from donor to acceptor residues. Such "scrambling" could result in inaccurate modification localization, potentially leading to misidentifications. Systematic studies presented herein provide mechanistic insights for the unusually high phosphate group rearrangements presented some time ago by Reid and coworkers (Proteomics 2013, 13 [6], 964-973). It is postulated here that a basic residue like histidine can play a key role in mediating the phosphate group transfer by deprotonating the serine acceptor site. The proposed mechanism is consistent with the observation that fast collisional activation by collision-cell CID and higher-energy collisional dissociation (HCD) can shut down rearrangement chemistry. Additionally, the rearrangement chemistry is highly dependent on the charge state of the peptide, mirroring previous studies that less rearrangement is observed under mobile proton conditions.

Gas-phase metal ion chelation investigated with IRMPD spectroscopy: A brief review of Robert Dunbar's contributions.

With the passing of Prof. Robert C. Dunbar on 31 October 2017, the field of ion chemistry lost one of its modern heroes. Throughout his career in mass spectrometry, two of his main research interests involved the interaction of trapped ions with electromagnetic radiation and the chelation motifs of metal ions with organic ligands. The focus of his early career was on the fundamental processes that take place in molecules upon ultraviolet and infrared excitation. From 2003 to 2017, his scientific interests shifted to more structural questions, notably to resolving the structures and binding motifs of metal ion chelation complexes by application of infrared photodissociation spectroscopy. These experiments were carried out during numerous visits to the (Free Electron Laser for Infrared eXperiments) (FELIX) facility in the Netherlands and were complemented by extensive theoretical investigations by Rob. As a tribute to our friend, we present in this contribution a brief review of this work.

Influence of Experimental Conditions on the Ratio of 25-Hydroxyvitamin D3 Conformers for Validating a Liquid Chromatography/Ion Mobility-Mass Spectrometry Method for Routine Quantitation.

The utility of adding ion mobility (IM) to liquid chromatography/mass spectrometry (LC/MS) for quantitation of 25-hydroxyvitamin D3 (25OHD3) is evaluated. Sodiated 25OHD3 ions adopt both closed and open conformations, whereas the stereoisomer 3-epi-25-hydroxyvitamin D (epi25OHD), when sodiated, adopts only a closed gas-phase conformation. The unique open conformation for sodiated 25OHD3 permits unambiguous quantitation. Nonetheless, the ratio of open versus closed gas-phase conformations for sodiated 25OHD3 can vary with instrumental conditions; conversion from the open to the closed conformer is attributed to radio frequency (rf) heating within the ion accumulation trap. Ion heating becomes significant when space charge in the trap forces ions to larger radii where the rf field is higher. To avoid biasing quantitation, an isotopically labeled internal standard must be used to account for changes in conformer ratio. Thirty-three serum extract samples were evaluated by LC/IM-MS and were found to not be biased by changes in ion conformer ratios, permitting reliable quantitation of 25OHD3 without interference from the epimer.

Operation and Performance of a Mass-Selective Cryogenic Linear Ion Trap.

We report on the performance of a cryogenic 2D linear ion trap (cryoLIT) that is shown to be mass-selective in the temperature range of 17-295 K. As the cryoLIT is cooled, the ejection voltages during the mass instability scan decrease, which results in an effective mass shift to lower m/z relative to room temperature. This is attributed to a decrease in trap radius caused by thermal contraction. Additionally, the cryoLIT generates reproducible mass spectra from day-to-day, and is capable of performing stored waveform inverse Fourier transform (SWIFT) mass isolation of fragile N2-tagged ions for the purpose of background-free infrared dissociation spectroscopy. Graphical Abstract ᅟ.

Probing the Structures of Solvent-Complexed Ions Formed in Electrospray Ionization Using Cryogenic Infrared Photodissociation Spectroscopy.

The gas-phase infrared photodissociation (IRPD) spectra of solvent-tagged small biomolecules are studied in a cryogenic ion trap at 17 K. In this study para-aminobenzoic acid (PABA) and tyramine molecules are noncovalently tagged with water or acetonitrile in the electrospray ionization (ESI) source. The complexes are then cooled in the cryogenic trap prior to spectroscopic measurements. These molecules provide two putative sites for solvent attachment: the protonated amine (NH3+) and the OH groups. Comparisons of the experimental IR spectra to theoretical spectra obtained with density functional theory show that the NH3+ site is mainly favored. Evidence for the formation of both NH3-bound and OH-bound conformers is found only in tyramine, despite having similar solution- and gas-phase energetics to that of PABA. Since the structures cannot interconvert in the gas phase, this suggests an isomerization during the electrospray process.

Radical Rearrangement Chemistry in Ultraviolet Photodissociation of Iodotyrosine Systems: Insights from Metastable Dissociation, Infrared Ion Spectroscopy, and Reaction Pathway Calculations.

We report on the ultraviolet photodissociation (UVPD) chemistry of protonated tyrosine, iodotyrosine, and diiodotyrosine. Distonic loss of the iodine creates a high-energy radical at the aromatic ring that engages in hydrogen/proton rearrangement chemistry. Based on UVPD kinetics measurements, the appearance of this radical is coincident with the UV irradiation pulse (8 ns). Conversely, sequential UVPD product ions exhibit metastable decay on ca. 100 ns timescales. Infrared ion spectroscopy is capable of confirming putative structures of the rearrangement products as proton transfers from the imine and ß-carbon hydrogens. Potential energy surfaces for the various reaction pathways indicate that the rearrangement chemistry is highly complex, compatible with a cascade of rearrangements, and that there is no preferred rearrangement pathway even in small molecular systems like these. Graphical Abstract.

Infrared ion spectroscopy: an analytical tool for the study of metabolites.

Vibrational ion spectroscopy techniques coupled with mass spectrometry are applied to standard metabolites as a proof-of-principle demonstration for the structural identification of unknown metabolites. The traditional room temperature infrared multiple photon dissociation (IRMPD) spectroscopy technique is shown to differentiate chemical moieties in isobaric and isomeric variants. These results are compared to infrared spectra of cryogenically cooled analyte ions, showing enhanced spectral resolution, and thus also improved differentiation between closely related molecules, such as isomers. The cryogenic spectroscopy is effected in a recently developed mass-selective cryogenic linear ion trap, which is capable of high sensitivity and the ability to measure the IR spectra of multiple analytes simultaneously.

Effects of ESI conditions on kinetic trapping of the solution-phase protonation isomer of p-aminobenzoic acid in the gas phase.

The effects of electrospray ionization (ESI) solvent and source temperature on the relative abundance of the preferred solution-phase (N-protonated; i.e. amine) versus preferred gas-phase (O-protonated; i.e., acid) isomers of p-aminobenzoic acid (PABA) were investigated. When PABA was electrosprayed from protic solvents (i.e., methanol/water), the infrared multiple photon dissociation (IRMPD) spectrum recorded was consistent with that for O-protonation, according to both calculations and previous studies. When aprotic solvent (i.e., acetonitrile) was used, a different spectrum was recorded and was assigned to the N-protonated isomer. As the amine is the preferred protonation site in solution, this suggests that an isomerization takes place under certain conditions. Photodissociation at the diagnostic band for the O-protonated isomer (NH2 stretching mode) was used to quantify the relative contributions of each isomer to ion signal as a function of ESI conditions. For mixtures of methanol and acetonitrile, the relative contribution of the O-protonated gas-phase structure increased as a function of methanol content. Yet, substituting methanol for water resulted in a marked decrease of isomerization to the O-protonated structure. The source temperature (i.e., temperature of a heated desolvation capillary) was found to play a key role in determining the extent of isomerization, with higher temperatures yielding increased presence of gas-phase structures. These results are consistent with a protic bridge mechanism, in which the ESI droplet temperatures, dependent on endothermic desolvation and radiative heating from the capillary, may determine the isomerization yield.

Infrared ion spectroscopy inside a mass-selective cryogenic 2D linear ion trap.

We demonstrate operation of the first cryogenic 2D linear ion trap (LIT) with mass-selective capabilities. This trap presents a number of advantages for infrared ion "action" spectroscopy studies, particularly those employing the "tagging/messenger" spectroscopy approach. The high trapping efficiencies, trapping capacities, and low detection limits make 2D LITs a highly suitable choice for low-concentration analytes from scarce biological samples. In our trap, ions can be cooled down to cryogenic temperatures to achieve higher-resolution infrared spectra, and individual ions can be mass selected prior to irradiation for a background-free photodissociation scheme. Conveniently, multiple tagged analyte ions can be mass isolated and efficiently irradiated in the same experiment, allowing their infrared spectra to be recorded in parallel. This multiplexed approach is critical in terms of increasing the duty cycle of infrared ion spectroscopy, which is currently a key weakness of the technique. The compact design of this instrument, coupled with powerful mass selection capabilities, set the stage for making cryogenic infrared ion spectroscopy viable as a bioanalytical tool in small molecule identification.

Supercharging Proteins: How Many Charges Can a Protein Carry?

Very highly charged proteins, so-called "supercharged" ions, can lose (excess) protons to background gases like N2 . It is remarkable that such extremely acidic species can be generated in electrospray ionization, in the presence of not just N2 but also much higher-basicity solvents. What mechanism(s) can explain such high charging, and what is the ultimate limit?

Amine vs. carboxylic acid protonation in ortho-, meta-, and para-aminobenzoic acid: An IRMPD spectroscopy study.

Infrared multiple photon dissociation (IRMPD) spectroscopy and computational chemistry are applied to the ortho-, meta-, and para- positional isomers of aminobenzoic acid to investigate whether the amine or the carboxylic acid are the favored sites of proton attachment in the gas phase. The NH and OH stretching modes yield distinct patterns that establish the carboxylic acid as the site of protonation in para-aminobenzoic acid, as opposed to the amine group in ortho- and meta-aminobenzoic acid, in agreement with computed thermochemistries. The trends for para- and meta-substitutions can be rationalized simplistically by inductive effects and resonant stabilization, and will be discussed in light of computed charge distributions based from electrostatic potentials. In ortho-aminobenzoic acid, the close proximity of the amine and acid groups allow a simultaneous interaction of the proton with both groups, thus stabilizing and delocalizing the charge more effectively, and compensating for some of the resonance stabilization effects.

Vibrational Signatures of Isomeric Lithiated N-acetyl-D-hexosamines by Gas-Phase Infrared Multiple-Photon Dissociation (IRMPD) Spectroscopy.

Three lithiated N-acetyl-D-hexosamine (HexNAc) isomers, N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GalNAc), and N-acetyl-D-mannosamine (ManNAc) are investigated as model monosaccharide derivatives by gas-phase infrared multiple-photon dissociation (IRMPD) spectroscopy. The hydrogen stretching region, which is attributed to OH and NH stretching modes, reveals some distinguishing spectral features of the lithium-adducted complexes that are useful in terms of differentiating these isomers. In order to understand the effect of lithium coordination on saccharide structure, and therefore anomericity, chair configuration, and hydrogen bonding networks, the conformational preferences of lithiated GlcNAc, GalNAc, and ManNAc are studied by comparing the experimental measurements with density functional theory (DFT) calculations. The experimental results of lithiated GlcNAc and GalNAc show a good match to the theoretical spectra of low-energy structures adopting a 4 C 1 chair conformation, consistent with this motif being the dominant conformation in condensed-phase monosaccharides. The epimerization effect upon going to lithiated ManNAc is significant, as in this case the 1 C 4 chair conformers give a more compelling match with the experimental results, consistent with their lower calculated energies. A contrasting computational study of these monosaccharides in their neutral form suggests that the lithium cation coordination with Lewis base oxygens can play a key role in favoring particular structural motifs (e.g., a 4 C 1 versus 1 C 4 ) and disrupting hydrogen bond networks, thus exhibiting specific IR spectral features between these closely related lithium-chelated complexes. Graphical Abstract ᅟ.

Making Mass Spectrometry See the Light: The Promises and Challenges of Cryogenic Infrared Ion Spectroscopy as a Bioanalytical Technique.

The detailed chemical information contained in the vibrational spectrum of a cryogenically cooled analyte ion would, in principle, make infrared (IR) ion spectroscopy a gold standard technique for molecular identification in mass spectrometry. Despite this immense potential, there are considerable challenges in both instrumentation and methodology to overcome before the technique is analytically useful. Here, we discuss the promise of IR ion spectroscopy for small molecule analysis in the context of metabolite identification. Experimental strategies to address sensitivity constraints, poor overall duty cycle, and speed of the experiment are intimately tied to the development of a mass-selective cryogenic trap. Therefore, the most likely avenues for success, in the authors' opinion, are presented here, alongside alternative approaches and some thoughts on data interpretation.

H2SO4 and SO3 transfer reactions in a sulfopeptide-basic peptide complex.

We report on the intermolecular transfer of sulfuric acid (H2SO4) and sulfur trioxide (SO3) from an acidic sulfopeptide (sSE) to a basic peptide (R3); this is achieved by subjecting a noncovalent complex of sSE + R3 to collisional activation in a quadrupole ion trap. The product ions resulting from the sulfo-group transfers were characterized by MS(3) experiments. Peak assignments were additionally supported by isotope-labeling and energy-resolved collision induced dissiciation (CID) experiments. The observed reactions and their potential implications for proteomics and post-translational modification discovery experiments are discussed.

Peptide fragmentation products in mass spectrometry probed by infrared spectroscopy.

Vibrational spectroscopy offers detailed insights, by virtue of diagnostic infrared bands, into the chemical structures and moieties which are formed during peptide fragmentation inside mass spectrometers. Over the past few years, IRMPD spectroscopy has led to a greatly improved understanding of the chemistry that takes place during collision-induced dissociation (CID) of protonated peptides. For instance, the rearrangement chemistry of b- and a-type ions, which is relevant in sequence scrambling pathways, has been directly confirmed with the technique. In this chapter, we provide a brief background on peptide fragmentation chemistry, and give an overview of areas where vibrational spectroscopy has been successfully implemented, such as CID of protonated and de-protonated peptides. We also discuss the potential of the technique for elucidating lesser-studied radical dissociation processes, such as electron capture dissociation (ECD), electron transfer dissociation (ETD), and laser photodissociation.

Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4.

This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin-trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion, both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping are similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein.

Linkage and anomeric differentiation in trisaccharides by sequential fragmentation and variable-wavelength infrared photodissociation.

Carbohydrates and their derivatives play important roles in biological systems, but their isomeric heterogeneity also presents a considerable challenge for analytical techniques. Here, a stepwise approach using infrared multiple-photon dissociation (IRMPD) via a tunable CO2 laser (9.2-10.7 µm) was employed to characterize isomeric variants of glucose-based trisaccharides. After the deprotonated trisaccharides were trapped and fragmented to disaccharide C2 fragments in a Fourier transform ion cyclotron resonance (FTICR) cell, a further variable-wavelength infrared irradiation of the C2 ion produced wavelength-dependent dissociation patterns that are represented as heat maps. The photodissociation patterns of these C2 fragments are shown to be strikingly similar to the photodissociation patterns of disaccharides with identical glycosidic bonds. Conversely, the photodissociation patterns of different glycosidic linkages exhibit considerable differences. On the basis of these results, the linkage position and anomericity of glycosidic bonds of disaccharide units in trisaccharides can be systematically differentiated and identified, providing a promising approach to characterize the structures of isomeric oligosaccharides.

Reaction of the C3(X(1)Σg (+)) carbon cluster with H2S(X(1)A1), hydrogen sulfide: photon-induced formation of C3S, tricarbon sulfur.

In this paper we report on the neutral-neutral reaction of the C3 carbon cluster with H2S in solid inert argon at 12 K, conditions that mimic, in part, the surfaces of interstellar grains. In the first step of the reaction, a C3•H2S complex is formed via an almost barrierless entrance addition mechanism. This complex, stabilized by an estimated 7.45 kJ/mol (CCSD(T)/aug-cc-pVTZ//B3LYP/6-311++G(d,p) level), is formed by the interaction of a terminal carbon of C3 with a hydrogen in H2S. This con-covalent complex displays a band at 2044.1 cm(-1) observed via Fourier transform infrared absorption spectroscopy. With the help of the MP2/aug-ccpVDZ level method, this band is assigned to the CC asymmetric vibration mode. When the complex is exposed to UV-visible photons (hν < 5.5 eV) the tricarbon sulfur C3S molecule is identified, based on the appearance of a characteristic CC stretching band at 2047.5 cm(-1). Calculated ground-state potential energy surfaces also confirm the concomitant formation of molecular H2. This facile reaction pathway involves an attainable transition state of 174.4 kJ/mol. Conversely, competing lower-energy reaction pathways that would lead to the generation of H2C3S (propadienethione), or C2H2 (acetylene) and CS, involve much more complex, multi-stage pathways, and are not observed experimentally.

Differentiating sulfopeptide and phosphopeptide ions via resonant infrared photodissociation.

The post-translational modifications sulfation and phosphorylation pose special challenges to mass spectral analysis due to their isobaric nature and their lability in the gas phase, as both types of peptides dissociate through similar channels upon collisional activation. Here, we present resonant infrared photodissociation based on diagnostic sulfate and phosphate OH stretches, as a means to differentiate sulfated from phosphorylated peptides within the framework of a mass spectrometry platform. The approach is demonstrated for a number of tyrosine-containing peptides, ranging from dipeptides (YG, pYG, and sYG) over tripeptides (GYR, GpYR, and GsYR), to more biologically relevant enkephalin peptides (YGGFL, pYGGFL, and sYGGFL). In all cases, the diagnostic ranges for sulfate OH stretches are established as 3580-3600 cm(-1) and can thus be distinguished from other characteristic hydrogen stretches, such as carboxylic acid OH, alcohol OH, and phosphate OH stretches.