Collision-induced Decomposition Research Articles

Study of the isomeric Maillard degradants, glycosylamine and Amadori rearrangement products, and their differentiation via MS2 fingerprinting from collision-induced decomposition of protonated ions.

The focus of this work was to study glycosylamine and Amadori rearrangement products (ARPs), the two major degradants in the Maillard reactions of pharmaceutical interest, and utilize their MS2 fingerprints by liquid chromatography/high-resolution tandem mass spectrometry (LC/HRMS2 ) to quickly distinguish the two isomeric degradants. These two types of degradants are frequently encountered in the compatibility and stability studies of drug products containing primary or secondary amine active pharmaceutical ingredients (APIs), which are formulated with excipients consisting of reducing sugar functionalities. Vortioxetine was employed as the primary model compound to react with lactose to obtain the glycosylamine and ARP degradants of the Maillard reaction, and their MS2 spectra (MS2 fingerprints) were obtained by LC/MS2 . Subsequently, the two degradants were isolated via preparative HPLC and their structures were confirmed by one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) determination. The MS2 fingerprints of the two degradants display significantly different profiles, despite the fact that many common fragments are observed. Specifically, protonated glycosylamine shows a prominent characteristic fragment of [Mvort + C2 H3 O]+ at m/z 341 (Mvort is the vortioxetine core), while protonated ARP shows a prominent characteristic fragment of [Mvort + CH]+ at m/z 311. Further study of the Maillard reactions between several other structurally diverse primary/secondary amines and lactose produced similar patterns. The study suggests that the characteristic MS2 fragment peaks and their ratios may be used to differentiate the glycosylamine and ARP degradants, the two isomeric degradants of the Maillard reaction, which are commonly encountered in finished dosage forms of pharmaceutical products containing primary and secondary amine APIs.

Center-of-Mass iso-Energetic Collision-Induced Decomposition in Tandem Triple Quadrupole Mass Spectrometry.

Two scan modes of the triple quadrupole tandem mass spectrometer, namely Collision Induced Dissociation Precursor Ion scan and Neutral Loss scan, allow selectively pinpointing, in a complex mixture, compounds that feature specific chemical groups, which yield characteristic fragment ions or are lost as distinctive neutral fragments. This feature of the triple quadrupole tandem mass spectrometer allows the non-target screening of mixtures for classes of components. The effective (center-of-mass) energy to achieve specific fragmentation depends on the inter-quadrupole voltage (laboratory-frame collision energy) and on the masses of the precursor molecular ion and of the collision gas, through a non-linear relationship. Thus, in a class of homologous compounds, precursor ions activated at the same laboratory-frame collision energy face different center-of-mass collision energy, and therefore the same fragmentation channel operates with different degrees of efficiency. This article reports a linear equation to calculate the laboratory-frame collision energy necessary to operate Collision-Induced Dissociation at the same center-of-mass energy on closely related compounds with different molecular mass. A routine triple quadrupole tandem mass spectrometer can operate this novel feature (iso-energetic collision-induced dissociation scan; i-CID) to analyze mixtures of endogenous metabolites by Precursor Ion and Neutral Loss scans. The latter experiment also entails the hitherto unprecedented synchronized scanning of all three quadrupoles of the triple quadrupole tandem mass spectrometer. To exemplify the application of this technique, this article shows two proof-of-principle approaches to the determination of biological mixtures, one by Precursor Ion analysis on alpha amino acid derivatized with a popular chromophore, and the other on modified nucleosides with a Neutral Fragment Loss scan.

Tandem mass spectrometry of novel ether-linked phospholipid analogs of anionic pulmonary surfactant phospholipids.

Structural analogs of the bioactive lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were synthesized with a xylitol polar head group and both diacyl and diether radyl groups. Mass spectral characterization of xylitol phospholipids (PX) was carried out using collisional activation and high-resolution mass measurements of positive molecular ion species and compared with the phosphatidylglycerol (PG) analogs. PX were synthesized using a transphosphatidylation reaction catalyzed by phospholipase D and purified by high-performance liquid chromatography (HPLC). Compounds were subjected to electrospray ionization and collision-induced dissociation (CID) was performed using a tandem quadrupole mass spectrometer to generate positive and negative molecular ions. Diether phospholipids were additionally analyzed by high-resolution mass spectrometry as protonated and sodiated molecular species in positive ion mode. Ester-linked PX analogs behaved similarly to PG after collisional activation of [M-H]- . The product ions formed by CID of the diether PG and PX negative ions only revealed information about the head group with no information about the aliphatic chains. In contrast, CID of protonated and sodiated diether phospholipid positive ions revealed reactions corresponding to cleavage of the ether chain, likely occurring by charge-driven reaction mechanisms. Novel PX analogs with diacyl and diether radyl substituents of the glycerol backbone were characterized by tandem mass spectrometry. These unique diether phospholipid analogs enabled exploration of ether cleavage reactions of the positive molecular ion species resulting from collision-induced decomposition. Copyright © 2016 John Wiley & Sons, Ltd.

Travelling-wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N-glycans.

Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1-acid glycoprotein and thyroglobulin were measured with a travelling-wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man3 GlcNAc3 from chicken ovalbumin and Man3 GlcNAc3 Fuc1 from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision-induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high-mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high-mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility-m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons, Ltd.

Kinetics analysis of thermal decomposition of ammonium dinitramide (ADN)

Kinetics analyses were performed on the thermal decomposition of ammonium dinitramide (ADN) using thermogravimetry-differential thermal analysis–mass spectrometry–infrared spectroscopy (TG-DTA–MS–IR). The main evolved gases were determined to be NH3, H2O, N2, NO, N2O, and NO2. The apparent activation energies of the exothermic, mass-change and gas-evolving reactions were analyzed on the basis of Friedman methods. The apparent activation energy of evolving N2 has the same value as that of evolving H2O since they occur by the same mechanism. A Friedman plot obtained from the DTA data has a curve similar to those obtained from N2 and H2O. The reaction that generated N2 and H2O plays an important role in the exothermic reaction in the decomposition of ADN. The activation energy for the N2O evolution reaction has a range of approximately 120–152 kJ mol−1 with reaction progress values between 0.1 and 0.9. Quantum chemistry calculations revealed that the total energy barrier of dinitramic acid unimolecular decomposition and ammonium-dinitramic ions collision-induced decomposition is 149.9–156.0 and 160.6 kJ mol−1, respectively. These values are reasonable compared with the experimental value of 152 kJ mol−1.

Improved Steroids Detection and Evidence for Their Regiospecific Decompositions Using Anion Attachment Mass Spectrometry

Nonpolar anabolic steroids are doping agents that typically do not provide strong signals by electrospray ionization-mass spectrometry (ESI-MS) owing especially to the low polarity of the functional groups present. We have investigated the addition of anions, in ammonium salt form, to anabolic steroid samples as ionization enhancers and have confirmed that lower instrumental limits of detection (as low as 10 ng/mL for fluoxymesterone-M) are obtained by fluoride anion attachment mass spectrometry, as compared to ESI(+)/(-) or atmospheric pressure photoionization (APPI)(+). Moreover, collision-induced decomposition (CID) spectra of precursor fluoride adducts of the bifunctional steroid "reduced pregnenolone" (containing two hydroxyl groups) and its d4-analogue provide evidence of regiospecific decompositions after attachment of fluoride anion to a specific hydroxyl group of the steroid. This type of charting of specific CID reaction pathways can offer value to selected reaction monitoring experiments (SRM) as it may result in a gain in selectivity in detection as well as in improvements in quantification.

Unraveling the architecture of plasma polymers by electrospray tandem mass spectrometry

This feature article presents the implementation of a controlled chemical degradation (namely ethanolysis) combined with electrospray tandem mass spectrometry (ESI-MS/MS) as an original analytical strategy to gain insights into the molecular composition of insoluble plasma polymers deposited from hexamethyldisiloxane (HMDSO). Dissociation rules were established for poly(dimethylsiloxane) standards (PDMS) holding trimethylsilyl and/or hydroxy and/or ethoxy end-groups, expected to be found as ethanolysis products released after the cleavage of bonds specifically at cross-linking points of the solid plasma polymer network. Mechanistic investigations were performed on the collision-induced decomposition (CID) of ammonium adducts of the selected standards to derive diagnostic ions typical of a given chain termination. Based on the so-listed diagnostic MS/MS features, the MS/MS analysis of the ethanolyzed samples allowed their structures to be deduced and the architecture of the original plasma polymers to be reconstructed.

Prostaglandin Extraction and Analysis in <em>Caenorhabditis elegans</em>

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 α-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in <em>Caenorhabditis elegans</em>

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 α-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Testing an alternative search algorithm for compound identification with the ‘Wiley Registry of Tandem Mass Spectral Data, MSforID’

A tandem mass spectral database system consists of a library of reference spectra and a search program. State-of-the-art search programs show a high tolerance for variability in compound-specific fragmentation patterns produced by collision-induced decomposition and enable sensitive and specific 'identity search'. In this communication, performance characteristics of two search algorithms combined with the 'Wiley Registry of Tandem Mass Spectral Data, MSforID' (Wiley Registry MSMS, John Wiley and Sons, Hoboken, NJ, USA) were evaluated. The search algorithms tested were the MSMS search algorithm implemented in the NIST MS Search program 2.0g (NIST, Gaithersburg, MD, USA) and the MSforID algorithm (John Wiley and Sons, Hoboken, NJ, USA). Sample spectra were acquired on different instruments and, thus, covered a broad range of possible experimental conditions or were generated in silico. For each algorithm, more than 30,000 matches were performed. Statistical evaluation of the library search results revealed that principally both search algorithms can be combined with the Wiley Registry MSMS to create a reliable identification tool. It appears, however, that a higher degree of spectral similarity is necessary to obtain a correct match with the NIST MS Search program. This characteristic of the NIST MS Search program has a positive effect on specificity as it helps to avoid false positive matches (type I errors), but reduces sensitivity. Thus, particularly with sample spectra acquired on instruments differing in their setup from tandem-in-space type fragmentation, a comparably higher number of false negative matches (type II errors) were observed by searching the Wiley Registry MSMS.

Stability of hydrated Ca2+ clusters studied by energy and pressure resolved collision-induced decomposition

Stability of hydrated Ca2+ clusters studied by energy and pressure resolved collision-induced decomposition

Silsesquioxanes as molecular analogues of single-site heterogeneous catalysts

We discuss herein selected examples of metal complexes of polyhedral oligosilses-quioxanes (POSSs) as models of single-site heterogeneous surface catalysts. The utility of these compounds as such models is illustrated when employed as analogues of single-site titanium species supported on a silica surface. Deep insights into structure–functionality relationships can be gained. In particular, it was possible to probe the relationship between accessibility of the reactive centre and turnover frequencies in a manner that is impossible for a purely heterogeneous catalyst. We also report that the partially dehydroxylated SiO 2 surface alone is an effective radical polymerization initiation catalyst. This surface reactivity is modelled by the solution reactions between the olefin substrate and two POSSs, the completely condensed triganol prism, Si 6 Cy 6 O 9 ( a6b0 , Cy = Cyclohexyl, C 6 H 11 ), and the incompletely condensed partial cube, Si 7 Cy 7 O 9 (OH) 3 ( a7b3 ). The former, with six-membered Si 3 O 3 rings, is a catalyst. The latter, without this feature, is not. Similar reactivity discrimination is observed in the gas phase reactions of these POSSs with the olefin substrate, examined using atmospheric pressure chemical ionization- and collision-induced decomposition spectroscopies. Silsesquioxane a6b0 , containing Si 3 O 3 rings, reacts with the olefin, forming grafted olefin monomers and dimers, while this reactivity is not observed with silsesquioxane a7b3 .

1,12-Diazaperylene and 2,11-dialkylated-1,12-diazaperylene iridium(iii) complexes [Ir(C^N)2(N^N)]PF6: new supramolecular assemblies

A series of new monocationic iridium(III) complexes [Ir(C^N)(2)(N^N)]PF(6) with "large-surface"α,α'-diimin ligands N^N (dap = 1,12-diazaperylene, dmedap = 2,11-dimethyl-1,12-diazaperylene, dipdap = 2,11-diisopropyl-1,12-diazaperylene) and different cyclometalating ligands C^N (piq = 1-phenylisoquinoline, bzq = benzo[h]quinoline, ppz = 1-phenylpyrazole, thpy = 2-(2-thienyl)pyridine, ppy = 2-phenylpyridine, meppy = 2-(4-methylphenyl)pyridine, dfppy = 2-(2,4-difluorophenyl)pyridine) were synthesized. The solid structures of the complexes [Ir(piq)(2)(dap)]PF(6), [Ir(bzq)(2)(dap)]PF(6), [Ir(ppy)(2)(dipdap)]PF(6), [Ir(piq)(2)(dmedap)]PF(6), [Ir(ppy)(2)(dap)]PF(6) and [Ir(ppz)(2)(dap)]PF(6) are reported. In [Ir(piq)(2)(dap)]PF(6), the dap ligand and one of the piq ligands of each cationic complex are involved in π-π stacking interactions forming supramolecular channels running along the crystallographic c axis. In the crystalline [Ir(bzq)(2)(dap)]PF(6)π-π stacking interactions between the metal complexes lead to the formation of a 2D layer structure. In addition, CH-π interactions were found in all compounds, which are what stabilizes the solid structure. In particular, a significant number of them were found in [Ir(piq)(2)(dap)]PF(6) and [Ir(bzq)(2)(dap)]PF(6). The crystal structures of [Ir(ppy)(2)(dipdap)]PF(6) and [Ir(ppy)(2)(dmedap)]PF(6) are also presented, being the first examples of bis-cyclometalated iridium(iii) complexes with phenanthroline-type α,α'-diimin ligands bearing bulky alkyl groups in the neighbourhood of the N-donor atoms. These ligands implicate a distorted octahedral coordination geometry that in turn destabilized the Ir-N(N^N) bonds. The new iridium(iii) complexes are not luminescent. All compounds show an electrochemically irreversible anodic peak between 1.15 and 1.58 V, which is influenced by the different cyclometalated ligands. All of the new complexes show two reversible successive one-electron "large-surface" ligand-centred reductions around -0.70 V and -1.30 V. Electrospray ionisation mass spectrometry (ESI-MS) and collision induced decomposition (CID) measurements were used to investigate the stability of the new complexes. Thereby, the stability agreed well with the order of the Ir-N(N^N) bond lengths.

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape(TM) from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.

Stability of disubstituted copper complexes in the gas phase analyzed by electrospray ionization mass spectrometry

A series of nitrogen ligand (L)/copper complexes of the type [Cu(I)L](+), [Cu(II)L(X)](+) and [Cu(I)L(2)](+) (X = Cl(-), BF(4) (-), acac(-), CH(3)COO(-) and SO(3)CF(3) (-)) was studied in the gas phase by electrospray ionization mass spectrometry. The following ligands (L) were employed: 1,12-diazaperylene (dap), 1,1'-bisisoquinoline (bis), 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 2,11-disubstituted 1,12-diazaperylenes (dap), 3,3'-disubstituted 1,1'- bisisoquinoline (bis), 5,8-dimethoxy-substituted diazaperylene (meodap), 6,6'- dimethoxy-substituted bisisoquinoline (meobis) and 2,9-dimethyl-1,10-phenanthroline (dmphen). Collision-induced decomposition measurements were applied to evaluate the relative stabilities of the different copper complexes. The influence of the spatial arrangement of the ligands, of the type of substituents and of the counter ion of the copper salts employed for the complexation was examined. Correlations were found between the binding constants of the [ML(2)](+) complexes in solution and the relative stabilities of the analogous complexes in the gas phase. Furthermore, complexation with the ligands 2,11-dialkylated 1,12-diazaperylenes [alkyl = ethyl (dedap) and isopropyl (dipdap)] was studied in the solvents CH(3)OH and CH(3)CN.

Characterization of an exception to the ‘even‐electron rule’ upon low‐energy collision induced decomposition in negative ion electrospray tandem mass spectrometry

Electrospray-generated precursor ions usually follow the 'even-electron rule' and yield 'closed shell' fragment ions. We characterize an exception to the 'even-electron rule.' In negative ion electrospray mass spectrometry (ES-MS), 2-(ethoxymethoxy)-3-hydroxyphenol (2-hydroxyl protected pyrogallol) easily formed a deprotonated molecular ion (M-H)(-) at m/z 183. Upon low-energy collision induced decomposition (CID), the m/z 183 precursor yielded a radical ion at m/z 124 as the base peak. The radical anion at m/z 124 was still the major fragment at all tested collision energies between 0 and 50 eV (E(lab)). Supported by computational studies, the appearance of the radical anion at m/z 124 as the major product ion can be attributed to the combination of a low reverse activation barrier and resonance stabilization of the product ions. Furthermore, our data lead to the proposal of a novel alternative radical formation pathway in the protection group removal of pyrogallol.

Enhanced Collision-Induced Decomposition Efficiency and Unraveling of Fragmentation Pathways for Anionic Adducts of Brevetoxins in Negative Ion Electrospray Mass Spectrometry

Brevetoxins are a group of natural neurotoxins characterized by polyether ring systems that are found in the blooms of red tide algae. In a conventional water/organic solvent system, without any other additives, deprotonated molecules of brevetoxins do not appear in high abundance in negative mode electrospray mass spectrometry (ES-MS) due to lack of acidic functional groups; thus, they are not well-suited for collision-induced decomposition (CID) experiments in negative mode electrospray. In this study, several anions were tested for their abilities to form anionic adducts by mixing ammonium salts of these anions with brevetoxin-2 and brevetoxin-3. Under CID, [M + Cl](-), [M + Br](-), [M + OAc](-), [M + HCOO](-), and [M + NO(3)](-) adducts all produced only the respective anions in CID experiments and thus provided no structural information. In contrast, upon CID, both [M + F](-) and [M + HCO(3)](-) precursor adducts gave structurally informative fragment peaks that exhibited similarities to those of [M - H](-) ions, which indicated that the first step in gas-phase decomposition of these anionic adducts was loss of neutral HF or H(2)CO(3) molecules, respectively, leaving deprotonated brevetoxin ([M - H](-)) to undergo consecutive fragmentations. In comparison to bicarbonate, fluoride formed adducts in higher abundance and provided more fragment peaks, thus more structural information, in MS/MS experiments. It is therefore the anion of choice to study brevetoxins in negative mode electrospray mass spectrometry using the anion attachment approach. The detailed fragmentation mechanisms are discussed, and diagnostic product ions are proposed for the brevetoxin-2 side chain (m/z 95, 133, and 151), the brevetoxin-3 side chain (m/z 97, 135, and 153), and the type-B brevetoxin backbone (m/z 725, 739, 741, and 797). To test the developed methodology, a sample of brevetoxin-2 subjected to in vitro microsomal incubation was analyzed, and a reduction product of the substrate was confirmed to have the structure of brevetoxin-3.

Gas phase behavior of radical cations of perfluoroalkyl‐1,2,4‐triazines: an experimental and theoretical study

Electron ionization mass spectrometry and low-energy collision-induced decomposition reactions occurring in a tridimensional ion trap, together with density functional theory (DFT) calculations on neutrals, even- and odd-electron cations, have been used to study the gas-phase ion chemistry of a series of perfluoroalkyl-1,2,4-triazines. Loss of oxygen, due to thermal degradation occurring before ionization, likely involving the hydroxylamino group, has been observed. Compounds having a carbonyl group at position 6 of the triazine ring fragment in the source by elimination of NO followed by HF or CO. The decomposition pathways occurring due to CID experiments have shown interesting features depending on the nature and structure of precursor ions. Most of them involve elimination of endocyclic atoms, thereby producing contraction of the original six-membered ring or formation of acyclic structures. DFT (B3LYP/6-31G(d,p)) calculations have been used for evaluating structure, stability and properties of neutral and ionic species involved in gas-phase processes. In particular, it has been calculated that in the molecular ion the unpaired electron is mainly located on the exocyclic nitrogen, while the positive charge is on the C(6) carbon atom.

Identification of phosphatidylserylglutamate: a novel minor lipid in Escherichia coli

Advances in mass spectrometry have facilitated the identification of novel lipid structures. In this work, we fractionated the lipids of Escherichia coli B and analyzed the fractions using negative-ion electrospray ionization mass spectrometry to reveal unknown lipid structures. Analysis of a fraction eluting with high salt from DEAE cellulose revealed a series of ions not corresponding to any of the known lipids of E. coli. The ions, with m/z 861.5, 875.5, 887.5, 889.5, and 915.5, were analyzed using collision-induced dissociation mass spectrometry (MS/MS) and yielded related fragmentation patterns consistent with a novel diacylated glycerophospholipid. Product ions arising by neutral loss of 216 mass units were observed with all of the unknowns. A corresponding negative product ion was also observed at m/z 215.0. Additional ions at m/z 197.0, 171.0, 146.0, and 128.0 were used to propose the novel structure phosphatidylserylglutamate (PSE). The hypothesized structure was confirmed by comparison with the MS/MS spectrum of a synthetic standard. Normal phase liquid chromatography-mass spectrometry analysis further showed that the endogenous PSE and synthetic PSE eluted with the same retention times. PSE was also observed in the equivalent anion exchange fractions of total lipids extracted from the wild-type E. coli K-12 strain MG1655.

Mechanical Injury and Cytokines Cause Loss of Cartilage Integrity and Upregulate Proteins Associated with Catabolism, Immunity, Inflammation, and Repair

The objectives of this study were to perform a quantitative comparison of proteins released from cartilage explants in response to treatment with IL-1beta, TNF-alpha, or mechanical compression injury in vitro and to interpret this release in the context of anabolic-catabolic shifts known to occur in cartilage in response to these insults in vitro and their implications in vivo. Bovine calf cartilage explants from 6-12 animals were subjected to injurious compression, TNF-alpha (100 ng/ml), IL-1beta (10 ng/ml), or no treatment and cultured for 5 days in equal volumes of medium. The pooled medium from each of these four conditions was labeled with one of four iTRAQ labels and subjected to nano-2D-LC/MS/MS on a quadrupole time-of-flight instrument. Data were analysed by ProQuant for peptide identification and quantitation. k-means clustering and biological pathways analysis were used to identify proteins that may correlate with known cartilage phenotypic responses to such treatments. IL-1beta and TNF-alpha treatment caused a decrease in the synthesis of collagen subunits (p < 0.05) as well as increased release of aggrecan G2 and G3 domains to the medium (p < 0.05). MMP-1, MMP-3, MMP-9, and MMP-13 were significantly increased by all treatments compared with untreated samples (p < 0.10). Increased release of proteins involved in innate immunity and immune cell recruitment were noted following IL-1beta and TNF-alpha treatment, whereas increased release of intracellular proteins was seen most dramatically with mechanical compression injury. Proteins involved in insulin-like growth factor and TGF-beta superfamily pathway modulation showed changes in pro-anabolic pathways that may represent early repair signals. At the systems level, two principal components were sufficient to describe 97% of the covariance in the data. A strong correlation was noted between the proteins released in response to IL-1beta and TNF-alpha; in contrast, mechanical injury resulted in both similarities and unique differences in the groups of proteins released compared with cytokine treatment.