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.

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).

Influence of Experimental Conditions on the Ratio of 25-Hydroxyvitamin D 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 D (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.

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.

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.

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.

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).

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.

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.

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.

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 N . It is remarkable that such extremely acidic species can be generated in electrospray ionization, in the presence of not just N 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.

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.

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.

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.

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.

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.

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.

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.

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).

Structures of biomolecular ions in the gas phase probed by infrared light sources.

Infrared (IR) spectroscopy of biomolecular ions combines mass spectrometry's high sensitivity and ability to analyze complex mixtures with the enhanced structural information available from vibrational spectroscopy. IR spectroscopy is in principle well placed to distinguish isomers and allow chemical classification of unknown molecules. This review gives an outline of current instrumentation, spectroscopic approaches, and potential bottlenecks.

Screening for phosphorylated and nonphosphorylated peptides by infrared photodissociation spectroscopy.

We present an infrared laser-based mass spectrometric strategy to differentiate peptides that are phosphorylated (i.e., containing pS, pT, or pY) from those that are nonphosphorylated (i.

Metal cation binding to gas-phase pentaalanine: divalent ions restructure the complex.

Ion-neutral complexes of pentaalalanine with several singly- and doubly charged metal ions are examined using conformation analysis by infrared multiple photon dissociation (IRMPD) spectroscopy and density functional theory (DFT) computations. The infrared spectroscopy in the 1500-1800 cm(-1) region is found to be conformationally informative; in particular, the frequency of the C═O stretching mode of the terminal carboxyl group is diagnostic for hydrogen bonding of the terminal hydroxyl. The doubly charged alkaline earth metal ions (Ca(2+) and Ba(2+)) enforce a highly structured chelation shell around the metal ion, with six strongly bound Lewis-basic chelation sites, and no hydroxyl hydrogen bonding.

Crown complexation of protonated amino acids: influence on IRMPD spectra.

We report infrared multiple photon dissociation (IRMPD) spectra for a series of crown-adducted, protonated amino acids, generated by electrospray ionization. The tight chelation of 18-crown-6 on the protonated NH(3)(+) moiety results in a considerable red shift of the NH(3)(+) stretch modes, notably the antisymmetric NH(3)(+) stretch. This is rationalized by a distortion of the NH(3)(+) normal mode potential energy surface, as verified by quantum chemical calculations.

Defying entropy: forming large head-to-tail macrocycles in the gas phase.

Spectral fingerprints: Collision-induced dissociation (CID) of protonated peptides in the gas phase results in linear fragment ions with a five-membered oxazolone ring on their C-terminal side. Infrared spectroscopy confirms that smaller fragments adopt oxazolone structures. Conversely, in mid-sized and larger fragments an isomerization to "head-to-tail" macrocycles is observed (see picture).