High-energy Collision-induced Dissociation Research Articles

High-energy CID tandem TOF-MS of various types of precursor ions of selected diether phospholipids: Diagnostic known and unexpected fragmentation pathways

The fragmentation behavior of some selected synthetic (1,2-diphytanyl- and 1,2-dihexadecyl-glycerophosphatidylethanolamine, 1,2-diphytanyl- and 1,2-dihexadecyl-glycerophosphatidylcholine) as well as of one natural diether phospholipid (2,3-diphytanyl-glycerophosphatidylinositol), the latter obtained from extracts of the archaeon Sulfolobus acidocaldaricus, was described by negative- and positive-ion MALDI high-energy CID tandem time-flight mass spectrometry for the first time. In contrast to the fragmentation pathways of classical diester glycerophospholipids, whose fragmentation behavior is already well described, the investigated diether glycerophospholipids exhibited a very different fragmentation behavior. The [M–H]−-precursor ions (ethanolamine, inositol) showed abundant high-mass charge-remote site fragmentation of the alkyl chains with easy determination of all methyl branching points (if present). Corresponding low mass product ions elucidated the identity of the polar head group. In contrast, [M+H]+-precursor ions of ethanolamine derivatives showed unusual loss of H3PO4 directly from the precursor ion and McLafferty-like rearrangements of selected product ions differing between sn1-and sn2-substituents, [R1O+58]+ and [R2O+42]+ ions, respectively. No diagnostic low mass product ions or high mass charge-remote site fragmentations are observed. A yet undescribed rearrangement reaction for protonated diether phosphocholine derivates was found by an intramolecular transesterification rearrangement of the precursor ion forming protonated O-alkyl glycerophosphatidylcholine. Besides, high mass charge-remote site fragmentation of the alkyl chains was observed. High-energy CID-spectra of [M+Na]+-precursor ions showed only little fragmentation (ethanolamine, inositol) with abundant partial polar head group losses and low mass head group product ions. In contrast, the [M+Na]+-precursor ions of corresponding choline derivatives showed significant charge-remote site fragmentation of the alkyl chains and diagnostic low mass head group ions. In case of the ethanolamine derivatives the [M+2Na–H]+-precursor ions exhibited abundant polar head group losses and high mass charge-remote site fragmentation with diagnostic low mass head group product ions. The inositol derivative mainly yielded disodiated dehydrated inositol phosphate as product ions. Finally, two diether phospholipid-specific product ions, the newly described K- and L-type ions, are described for the first time for all lipid derivatives and their mechanism of formation is described in detail.

Identification of Unique Fragmentation Patterns of Fentanyl Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry.

The opioid crisis in the United States is being fueled by the rapid emergence of new fentanyl analogs and precursors that can elude traditional library-based screening methods, which require data from known reference compounds. Since reference compounds are unavailable for new fentanyl analogs, we examined if fentanyls (fentanyl + fentanyl analogs) could be identified in a reference-free manner using a combination of electrospray ionization (ESI), high-resolution ion mobility (IM) spectrometry, high-resolution mass spectrometry (MS), and higher-energy collision-induced dissociation (MS/MS). We analyzed a mixture containing nine fentanyls and W-15 (a structurally similar molecule) and found that the protonated forms of all fentanyls exhibited two baseline-separated IM distributions that produced different MS/MS patterns. Upon fragmentation, both IM distributions of all fentanyls produced two high intensity fragments, resulting from amine site cleavages. The higher mobility distributions of all fentanyls also produced several low intensity fragments, but surprisingly, these same fragments exhibited much greater intensities in the lower mobility distributions. This observation demonstrates that many fragments of fentanyls predominantly originate from one of two different gas-phase structures (suggestive of protomers). Furthermore, increasing the water concentration in the ESI solution increased the intensity of the lower mobility distribution relative to the higher mobility distribution, which further supports that fentanyls exist as two gas-phase protomers. Our observations on the IM and MS/MS properties of fentanyls can be exploited to positively differentiate fentanyls from other compounds without requiring reference libraries and will hopefully assist first responders and law enforcement in combating new and emerging fentanyls.

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2 O. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

Tandem Mass Spectrometry de novo Sequencing of the Skin Defense Peptides of the Central Slovenian Agile Frog Rana dalmatina.

Peptides released on frogs' skin in a stress situation represent their only weapon against micro-organisms and predators. Every species and even population of frog possesses its own peptidome being appropriate for their habitat. Skin peptides are considered potential pharmaceuticals, while the whole peptidome may be treated as a taxonomic characteristic of each particular population. Continuing the studies on frog peptides, here we report the peptidome composition of the Central Slovenian agile frog Rana dalmatina population. The detection and top-down de novo sequencing of the corresponding peptides was conducted exclusively by tandem mass spectrometry without using any chemical derivatization procedures. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), electron transfer dissociation (ETD) and combined MS3 method EThcD with stepwise increase of HCD energy were used for that purpose. MS/MS revealed the whole sequence of the detected peptides including differentiation between isomeric Leu/Ile, and the sequence portion hidden in the disulfide cycle. The array of the discovered peptide families (brevinins 1 and 2, melittin-related peptides (MRPs), temporins and bradykinin-related peptides (BRPs)) is quite similar to that of R. temporaria. Since the genome of this frog remains unknown, the obtained results were compared with the recently published transcriptome of R. dalmatina.

In-Source Decay MALDI and High-Energy Collision-Induced Dissociation Mass Spectrometry of Alkali Metal-Adducted Underivatized Oligosaccharides.

In-source decay (ISD) and high-energy collision-induced dissociation (HE-CID) were explored to provide structural information on alkali metal-adducted linear and stacked oligosaccharides (oligosaccharides with increased flexibility due to linkage type). These oligosaccharides include isomeric tetrasaccharides, maltoheptaose, and several human milk oligosaccharides (HMOs). Matrix-assisted laser desorption ionization (MALDI) ion production efficiency, as well as the product ion intensities, and the number of product ions formed in ISD and HE-CID of these oligosaccharides were influenced by the matrix, the ionic radius of the metal ion used for adduction, and the affinity of metal ions for specific functional groups in the oligosaccharides. 2,4,6-Trihydroxyacetophenone (THAP) was the best matrix for HE-CID of oligosaccharides, 4-dimethylaminobenzaldehyde (DMABA) worked best for ISD of tetrasaccharides and pentasaccharides, while 2,5-dihydroxybenzoic acid (DHB) was the best matrix for ISD and HE-CID of long chain oligosaccharides. In general, the number of product ions formed followed the trend Li+ > Na+ > K+ > Rb+ > Cs+, except for HMOs where Na+ ≥ Li+ > K+ > Rb+ > Cs+ occurred. The type of product ions formed and their intensities varied based on the position of the glycosidic bond linkage and the content of the monosaccharide. ISD and HE-CID produced diagnostic ions that could structurally differentiate isomers. Overall, HE-CID of alkali-metal adducted oligosaccharides produces intense glycosidic bond cleavages and low intensity cross-ring and internal cleavages. In contrast, ISD generates mainly cross-ring cleavages and internal cleavages at intensities higher than in HE-CID. In addition, ISD produced unique product ions that complement results from HE-CID.

A Dual-Gated Structures for Lossless Ion Manipulations-Ion Mobility Orbitrap Mass Spectrometry Platform for Combined Ultra-High-Resolution Molecular Analysis.

High-resolution ion mobility spectrometry-mass spectrometry (HR-IMS-MS) instruments have enormously advanced the ability to characterize complex biological mixtures. Unfortunately, HR-IMS and HR-MS measurements are typically performed independently due to mismatches in analysis time scales. Here, we overcome this limitation by using a dual-gated ion injection approach to couple an 11 m path length structures for lossless ion manipulations (SLIM) module to a Q-Exactive Plus Orbitrap MS platform. The dual-gate setup was implemented by placing one ion gate before the SLIM module and a second ion gate after the module. The dual-gated ion injection approach allowed the new SLIM-Orbitrap platform to simultaneously perform an 11 m SLIM separation, Orbitrap mass analysis using the highest selectable mass resolution setting (up to 140 k), and high-energy collision-induced dissociation (HCD) in ∼25 min over an m/z range of ∼1500 amu. The SLIM-Orbitrap platform was initially characterized using a mixture of standard phosphazene cations and demonstrated an average SLIM CCS resolving power (RpCCS) of ∼218 and an SLIM peak capacity of ∼156, while simultaneously obtaining high mass resolutions. SLIM-Orbitrap analysis with fragmentation was then performed on mixtures of standard peptides and two reverse peptides (SDGRG1+, GRGDS1+, and RpCCS = 305) to demonstrate the utility of combined HR-IMS-MS/MS measurements for peptide identification. Our new HR-IMS-MS/MS capability was further demonstrated by analyzing a complex lipid mixture and showcasing SLIM separations on isobaric lipids. This new SLIM-Orbitrap platform demonstrates a critical new capability for proteomics and lipidomics applications, and the high-resolution multimodal data obtained using this system establish the foundation for reference-free identification of unknown ion structures.

Integrating Effect-Directed Analysis and Chemically Indicative Mass Spectral Fragmentation to Screen for Toxic Organophosphorus Compounds.

Analytical chemists are often challenged to screen for bioactive compounds in complex matrices, sometimes without a priori knowledge of the exact compound of interest. Therefore, "flagging" techniques, highlighting common characteristics of bioactive compounds, are highly sought after. In this work, we demonstrate a double flagging method, where unknown organophosphorus acetylcholinesterase inhibitors are "flagged" out of a complex matrix by the presence of organophosphorus-indicative ions as well as their acetylcholinesterase inhibition. This is accomplished by flagging the LC chromatographic retention time of phosphorus-indicative ions using accurate mass high-energy in-source CID products, and the retention time of acetylcholinesterase inhibiting compounds using a parallel microfractionation-based bioassay. We successfully apply this method to screen VX, VM, and RVX nerve agents as well as methomyl, a carbamate pesticide, out of soil and whole blood samples at low μM to sub-μM concentrations. This methodology can be easily extended to diverse chemical families and biological activities of interest.

In-depth characterization of nitrogen heterocycles of petroleum by liquid chromatography-energy-resolved high resolution tandem mass spectrometry

With the increased attention to processing heavy crude oils, a detailed description of chemical composition is critical for the petroleum refining industry. The current analytical technique such as ultrahigh resolution mass spectrometry has been successfully applied for the molecular level characterization of complex petroleum fractions. But the structural characterization of heavy petroleum feedstock is still a great challenge. In this study, a novel in-depth characterization method of nitrogen heterocycles (N-heterocycles) in heavy petroleum mixtures was proposed by online liquid chromatography coupled with electrospray ionization high resolution energy-resolved mass spectrometry. A series of typical basic aromatic, neutral aromatic and naphtheno-aromatic nitrogen heterocyclic model compounds were synthesized to investigate energy-resolved fragmentation behaviors in high energy collision-induced dissociation at 10–100 eV. Energy-dependent fragmentation pathways were elucidated. Notably, characteristic double bond equivalent (DBE) versus carbon number distributions of N1 ions and all CH ions were discovered, which were closely related to their core structure. Then a workflow to assign core structures of alkyl-substituted N-heterocycles in petroleum was proposed and validated. The developed method was applied to investigate the structural isomers in feed and product vacuum gas oil (VGO) fractions. Core structural differences in feed VGO and subtle structural variations between feed and product VGOs were recognized. This work can distinguish structural isomers of N-heterocycles with the subtle difference in their core structure in heavy petroleum fractions based on global energy dimensional fragmentation characteristics.

A strategy integrating parent ions list-modified mass defect filtering-diagnostic product ions for rapid screening and systematic characterization of flavonoids in Scutellaria barbata using hybrid quadrupole-orbitrap high-resolution mass spectrometry

In this study, full scan (FS)-parent ions list (PIL)-higher energy collision induced dissociation (HCD)-MS/MS (FS-PIL-HCD-MS/MS) was used to acquire the chemical profile of flavonoids in Scutellaria barbata. Mass defect filtering (MDF) induced subtype classification and diagnostic product ions (DPIs) dominated structural confirmation were integrated into an effective strategy for the systematic screening and identification of the flavonoids. An in-house flavonoid MS database based on molecular design was established to construct a modified triangle MDF algorithm for progressive screening and subtype classification. The obtained results demonstrated that the modified MDF was capable of simplifying the workload in formula editing and subsequent screening process, and distinguishing different subtypes. The fragmentation behaviors of eleven reference standards were evaluated to obtain the MS2 fragmentation pathway and DPIs which can provide a criterion to eliminate false-positive results and judge the target flavonoids with the exact number and position of substituents for the first time. Structure confirmation was characterized by comparing with the reference substance, searching the database, and analyzing DPIs. To distinguish some isomers, ClogP (the calculated lipophilicity parameter) was adopted. As a result, 127 target flavonoids, including 30 flavone/flavonol aglycones, 10 flavanone/flavanonol aglycones, 49 flavone/flavonol monoglycosides, 16 flavanone/flavanonol monoglycosides, 21 flavone/flavonol diglycosides and 1 flavanone/flavanonol diglycoside, were ultimately identified or tentatively characterized based on the MS fragmentation pathway and DPIs analysis. This study provides a novel MDF method with improved subtype classification and develops a novel strategy for the progressive screening, subtype classification and systematic characterization of complex components in herbal medicines.

Oligosaccharide mapping analysis by HILIC-ESI-HCD-MS/MS for structural elucidation of fucoidan from sea cucumber Holothuria floridana

The elucidation of precise structure of fucoidan is essential for understanding their structure-function relationship and promoting the development of marine drugs. In this work, we firstly reported the oligosaccharide mapping of fucoidan from Holothuria floridana using a combination of hydrothermal depolymerization, hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray mass spectrometry (ESI-FTMS) and high energy collision-induced dissociation (HCD-MS/MS) and 2D NMR analysis. With careful selection of fully deprotonated molecular ions of fucoidan oligosaccharides and their NaBD4 reduced alditols, HILIC-ESI-HCD-MS/MS provided structurally relevant glycosidic product ions with no sulfate loss for definitive assignment of sequence and sulfation pattern of all the oligosaccharides and their isomers from dp2 to dp7 from hydrothermal depolymerization. The oligosaccharide mapping clarified the structure of fucoidan with various oligosaccharide domains with 2,4-di-O-sulfated and 2-O sulfated fucose residues.

O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins.

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized through high-resolution mass spectrometry. Following digestion with trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the GalNAc-type O-glycosylation pattern of S proteins, including O-glycosites and the six most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 255 intact O-glycopeptides composed of 50 peptides sequences and 43 O-glycosites were discovered by higher energy collision-induced dissociation (HCD), and three O-glycosites were confidently identified by electron transfer/higher energy collision-induced dissociation (EThcD) in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex (1). In contrast, in the human cell-expressed S protein S1 subunit, 407 intact O-glycopeptides composed of 34 peptides sequences and 30 O-glycosites were discovered by HCD, and 11 O-glycosites were unambiguously assigned by EThcD. However, the measurement of O-glycosylation occupancy hasn’t been made. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex (1)NeuAc (1) and HexNAc(1)Hex (1)NeuAc (2). Our results reveal that the SARS-CoV-2 S protein is an O-glycoprotein; the O-glycosites and O-glycan compositions vary with the host cell type. These comprehensive O-glycosylation landscapes of the S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.

Manual mass spectrometry de novo sequencing of the anionic host defense peptides of the Cuban Treefrog Osteopilus septentrionalis.

Host defense peptides accumulated in the skin glands of the animals constitute the basis of the adaptive and immune system of amphibians. The peptidome of the Cuban frog Osteopilus septentrionalis was established using tandem mass spectrometry as the best analytical tool to elucidate the sequence of these peptides. Manual interpretation of complementary collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), and electron transfer dissociation (ETD) tandem mass spectra recorded with an Orbitrap Elite mass spectrometer in liquid chromatography/mass spectrometry (LC/MS) mode was used to sequence the peptide components of the frog skin secretion, obtained by mild electrostimulation. Although the vast majority of amphibian peptides discovered so far are cationic, surprisingly only anionic peptides were identified in the skin secretion of the Cuban frog Osteopilus septentrionalis. Mass spectrometry allowed the sequences to be established of 16 representatives of new peptide families: septenins 1 and septenins 2. The highest sequence coverage when dealing with these anionic peptides was obtained with CID normalized collision energy 35 and HCD normalized collision energy 28. Mirror-symmetrical peptides are sequenced using N-terminal acetylation. Acetylated Ser is reliably distinguished from isomeric Glu by the loss of ketene from b-ions containing the corresponding residue. Calculations of the physicochemical and structural properties of the discovered anionic septenins 1 and 2 allowed the mechanism of their interaction with microbe cells to be postulated.

Selective screening for “unknown” phosphorous-containing compounds using high-resolution accurate-mass LC-MS

An analytical technique was developed for the detection of phosphorus-containing compounds, using high-resolution-accurate-mass (HRAM) LC-MS. This method was constructed as a “flagging” approach for chromatographic peaks of compounds suspected of containing phosphorus, using high-energy in-source CID fragmentation, in parallel with a Full-MS experiment. Selective peak picking is achieved by tracking the highly resolved low m/z product ions of phosphorus-containing products, indicative of several phosphate and phosphonate groups. The chemical formula is then specifically assigned by a simultaneous accurate mass measurement of the precursor ion, together with the confirmed presence of a phosphorus atom in the suspect peak. This new method is demonstrated with various phosphorus-containing compounds such as V-class chemical warfare agents, nerve agents’ degradation products, pesticides, organic phosphates and a phospholipid. For these compounds, the developed method was found to be effective at concentrations as low as 1 ng/mL, in environmental extracts of soil and in urine (in the case of the phospholipid), making it comparable to selective phosphorus detectors such as GC-FPD, but with a much broader scope for charged, polar, or larger compounds.

Identification and characterization of the chemical components of Iris tectorum Maxim. and evaluation of their nitric oxide inhibitory activity.

Iris tectorum Maxim. is a traditional medicinal herb that is commonly used to treat inflammatory conditions. The present study investigated the fragmentation patterns of isoflavone glycosides and their qualitative analysis. In addition, lipopolysaccharide (LPS)-induced RAW264.7 macrophages were used to evaluate the anti-inflammatory properties of I. tectorum Maxim. samples collected at different time points during the year. High-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (HPLC/QTOF-MS/MS) and HPLC with diode-array detection were employed for qualitative and quantitative analysis. The fragmentation patterns of the isoflavones were observed in negative electrospray ionization mode with collision-induced dissociation (CID). Their anti-inflammatory activity was assessed via nitric oxide (NO) production in LPS-treated RAW264.7 macrophages. A total of 15 chemical components were observed and tentatively identified using HPLC/QTOF-MS/MS. At low collision energy, the relative abundances of the aglycone radical anions Y0 - , [Y0  - H]-• , [Y0  - CH3 ]-• and [Y0  - H- CH2 ]-• were used for the structural characterization of tectoridin and tectorigenin-4'-O-β-D-glucoside. The radical ions [Y0  - CH3 ]-• and [Y0  - H - 2CH3 ]-• were also employed to differentiate between iristectorin A and iristectorin B based upon their high-energy CID spectra. Levels of 9.02 mg/g of tectoridin and 1.04 mg/g of tectorigenin were found in samples collected in June, which exhibited 69.7% NO inhibitory activity. The characteristic fragmentation patterns enabled us to reliably identify isoflavone glycosides. The results of the quantitative determination and NO inhibitory activity offer insight into the optimal I. tectorum Maxim. harvesting time.

Using electrospray ionization-tandem mass spectrometry to explore formation and gas-phase chemistry of silver nanoclusters generated from the reaction of silver salts with NaBH4 in the presence of bis(diphenylarsino)methane.

Electrospray ionization-mass spectrometry (ESI-MS) of mixtures of AgBF4 or AgNO3 with the capping ligand bis(diphenylarsino)methane ((Ph2 As)2 CH2 = dpam) in a solution of acetonitrile revealed the formation of the following cations: [Ag(CH3 CN)(dpam)]+ , [Ag(dpam)2 ]+ , [Ag2 (Cl)(dpam)2 ]+ , and [Ag3 (Cl)2 (dpam)3 ]+ . Addition of NaBH4 to these solutions results in the formation of the cluster cations [Ag2 (BH4 )(dpam)2 ]+ , [Ag2 (BH4 )(dpam)3 ]+ , [Ag3 (H)(BH4 )(dpam)3 ]+ , [Ag3 (BH4 )2 (dpam)3 ]+ , [Ag3 (H)(Cl)(dpam)3 ]+ , and [Ag3 (I)(BH4 )(dpam)3 ]+ , as established by ESI-MS. Use of NaBD4 confirmed that borohydride is the source of the hydride in these clusters. An Orbitrap Fusion LUMOS mass spectrometer was used to explore the gas-phase unimolecular chemistry of selected clusters via multistage mass spectrometry (MSn ) experiments employing low-energy collision-induced dissociation (CID) and high-energy collision-induced dissociation (HCD) experiments. The borohydride containing clusters fragment via two competing pathways: (i) ligand loss and (ii) B-H bond activation involving BH3 loss. Density functional theory (DFT) calculations were used to calculate the energetics of the optimized structures for all precursor ions, fragment ions, and neutrals and to estimate the reaction endothermicities. Generally, there is reasonable agreement between the most abundant product ion formed and the predicted endothermicity of the associated reaction channel. The DFT calculations predicted that the novel dimer [Ag2 (BH4 )(dpam)2 ]+ has a paddlewheel structure in which the dpam and BH4 - ligands bridge both silver centers.

Drug metabolite identification using ultrahigh-performance liquid chromatography-ultraviolet spectroscopy and parallelized scans on a tribrid Orbitrap mass spectrometer.

To capture all metabolites in metabolite identification studies, MS/MS information is required in both positive and negative ionization mode, usually involving several sample injections to gain all information about samples. A high-resolution and high mass accuracy quadrupole/linear trap/Orbitrap tribrid instrument was used to gain this information in a novel single injection 'capture-all' approach to metabolite identification. Diclofenac, a model compound, was incubated in human and rat hepatocytes. These incubated samples were run using an ultrahigh-performance liquid chromatography/ultraviolet (UHPLC-UV) system coupled to a Thermo Fusion tribrid mass spectrometer. Five parallel scans were used: positive and negative ion full scan, data-dependent MS/MS, both high energy dissociation and collision-induced dissociation, and data-independent all ion fragmentation (AIF) spectra were collected in positive and negative ion mode. Nine metabolites were identified; a metabolite observed in the UV trace, but not positive ion full scan MS, was detected in the same sample injection by negative ion full scan MS. This was identified as a sulphate metabolite, and the corresponding negative ion AIF allowed for some structural elucidation. The use of a photo-diode array (PDA) detector allowed for spectral assessment in case of changes in absorbance spectra, and the subsequent semi-quantification of metabolites. This method provided good-quality MS/MS data across the m/z range in both positive and negative ion mode. The addition of both negative ion full scan MS and negative ion MS/MS allowed for the detection and structural elucidation of metabolites not observed in positive ion mode. The use of the PDA detector allowed for the semi-quantification of metabolites.

Orbitrap mass spectrometry for monitoring the ganglioside pattern in human cerebellum development and aging.

We have developed here a superior approach based on high-resolution (HR) mass spectrometry (MS) for monitoring the changes occurring with development and aging in the composition and structure of cerebellar gangliosidome. The experiments were focused on the comparative screening and structural analysis of gangliosides expressed in fetal and aged cerebellum by Orbitrap MS with nanoelectrospray ionization (nanoESI) in the negative ion mode. The employed ultrahigh-resolution MS platform allowed the discrimination, without the need of previous separation, of 159 ions corresponding to 120 distinct species in the native ganglioside mixtures from fetal and aged cerebellar biopsies, many more than detected before, when MS platforms of lower resolution were employed. A number of gangliosides, in particular polysialylated belonging to GT, GQ, GP, and GS classes, modified by O-fucosylation, O-acetylation, or CH3 COO- were discovered here, for the first time in human cerebellum. These components, found differently expressed in fetal and aged tissues, indicated that the ganglioside profile in cerebellum is development stage- and age-specific. Following the fragmentation analysis by high-energy collision-induced dissociation (HCD) tandem MS (MS/MS), we have also observed that the intimate structure of certain compounds has not changed during the development and aging of the brain, an aspect which could open new directions in the investigation of ganglioside biomarkers in cerebellar tissue.

Decomposition of protonated ronidazole studied by low-energy and high-energy collision-induced dissociation and density functional theory.

Nitroimidazoles are important compounds in medicine, biology, and the food industry. The growing need for their structural assignment, as well as the need for the development of the detection and screening methods, provides the motivation to understand their fundamental properties and reactivity. Here, we investigated the decomposition of protonated ronidazole [Roni+H]+ in low-energy and high-energy collision-induced dissociation (CID) experiments. Quantum chemical calculations showed that the main fragmentation channels involve intramolecular proton transfer from nitroimidazole to its side chain followed by a release of NH2CO2H, which can proceed via two pathways involving transfer of H+ from (1) the N3 position via a barrier of TS2 of 0.97 eV, followed by the rupture of the C-O bond with a thermodynamic threshold of 2.40 eV; and (2) the -CH3 group via a higher barrier of 2.77 eV, but with a slightly lower thermodynamic threshold of 2.24 eV. Electrospray ionization of ronidazole using deuterated solvents showed that in low-energy CID, only pathway (1) proceeds, and in high-energy CID, both channels proceed with contributions of 81% and 19%. While both of the pathways are associated with small kinetic energy release of 10-23 meV, further release of the NO• radical has a KER value of 339 meV.

Sensitive β-substituted aliphatic Ile/Val-Xxx and aromatic Phe/Tyr/His-Xxx residues to form [a]+ ions in high energy CID of peptides

Residue-specific cleavages resulting in the formation of [a]+ ions of peptides have been examined using 20 keV high-energy (high-E) collision-induced dissociation (CID) with a tandem time-of-flight (TOF/TOF) instrument. High-E CID resulted in relatively sensitive cleavage at the Cα-C bond of β-substituted aliphatic Val/Ile-Xxx and aromatic Phe/Tyr/His-Xxx residues when an Arg residue was located on the N-terminal side. The sensitive aromatic Phe/Tyr/His residues and insensitive Gly residue in high-E CID are similar to other methods, such as ultraviolet photodissociation (UVPD) of [M+H]+, low-E CID of [M].+ and MALDI-ISD with a hydrogen-abstracting matrix. The properties of the Val/Ile residues are not necessarily common to the same methods. The high-sensitivity of Val/Ile and Phe/Tyr/His residues to high-E CID may be due to high-energy single collision between target gas and the bulky sidechains of these residues. It is suggested that from high-E CID and MALDI-ISD experiments the formation of [a]+ ions is essentially connected with the loss of atomic hydrogen from protonated peptides [M+H]+, and that high-E CID results in the loss of hydrogen from the backbone amide nitrogen rather than the site of the β-carbon of sidechains.

Higher-energy collision-induced dissociation for the quantification by liquid chromatography/tandem ion trap mass spectrometry of nitric oxide metabolites coming from S-nitroso-glutathione in an in vitro model of the intestinal barrier.

The potency of S-nitrosoglutathione (GSNO) as a nitric oxide (NO) donor to treat cardiovascular diseases (CVDs) has been highlighted in numerous studies. In order to study its bioavailability after oral administration, which represents the most convenient route for the chronic treatment of CVDs, it is essential to develop an analytical method permitting (i) the simultaneous measurement of GSNO metabolites, i.e. nitrite, S-nitrosothiols (RSNOs) and nitrate and (ii) to distinguish them from other sources (endogenous synthesis and diet). Exogenous GSNO was labeled with 15 N, and the GS15 NO metabolites after conversion into the nitrite ion were derivatized with 2,3-diaminonaphthalene. The resulting 2,3-naphthotriazole was quantified by liquid chromatography/tandem ion trap mass spectrometry (LC/ITMS/MS) in multiple reaction monitoring mode after Higher-energy Collision-induced Dissociation (HCD). Finally, the validated method was applied to an in vitro model of the intestinal barrier (monolayer of Caco-2 cells) to study GS15 NO intestinal permeability. A LC/ITMS/MS method based on an original transition (m/z 171 to 156) for sodium 15 N-nitrite, GS15 NO and sodium 15 N-nitrate measurements was validated, with recoveries of 100.8 ± 3.8, 98.0 ± 2.7 and 104.1 ± 3.3%, respectively. Intra- and inter-day variabilities were below 13.4 and 12.6%, and the limit of quantification reached 5 nM (signal over blank = 4). The permeability of labeled GS15 NO (10-100 μM) was evaluated by calculating its apparent permeability coefficient (Papp ). A quantitative LC/ITMS/MS method using HCD was developed for the first time to selectively monitor GS15 NO metabolites. The assay allowed evaluation of GS15 NO intestinal permeability and situated this drug candidate within the middle permeability class according to FDA guidelines. In addition, the present method has opened the perspective of a more fundamental work aiming at studying the fragmentation mechanism leading to the ion at m/z 156 in HCD tandem mass spectrometry in the presence of acetonitrile.