Identification of Barrenwort flavonoids by high-resolution tandem mass spectrometry

Fragmentation of the main Barrenwort flavonoids—icariin, icaritin, icarisides I and II, and epimedins A and B—is studied by tandem mass spectrometry. High-resolution mass spectra of positively charged ions of these compounds are obtained under the conditions of collision-induced dissociation. Characteristic fragment ions are determined, which ensured the classification of unknown compounds as Barrenwort flavonoids. Epimedin C was isolated from raw plant material by preparative liquid chromatography; its structure was confirmed by ¹H and ¹³C NMR spectra.     

Identification of degradation products of indigoids by tandem mass spectrometry

The study concerns identification of photodegradation products of indigotin, indirubin and isoindigo. Experimental methodology consists of degradation of standard solutions of indigoids in a solar box and analysis of samples taken at different aging time by using capillary high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometric and spectrophotometric detectors. Identification of the formed compounds was based on careful interpretation of the electrospray ionization MS/MS spectra. Apart from the well‐known degradation products of indigoids: isatin, isatoic anhydride and anthranilic acid, another seven species were also identified, and their proposed structures were confirmed by high‐resolution molecular masses measurements; according to the best knowledge of authors, they have not been reported so far. The obtained results formed the basis for postulating mechanism of the process. Moreover, the MRM (Multiple Reaction Monitoring) method was developed for the identification of natural dyes and their degradation products in textiles of historical value. Apart from such colorants as indigotin and flavonoids, also presence of degradation products of indigoids was confirmed. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of leucine-enkephalin radical oxidation products by liquid chromatography tandem mass spectrometry

The oxidation of the peptide leucine-enkephalin (YGGFL) induced by the hydroxyl radical (HO*), formed under Fenton-like conditions [Cu (II)/H(2)O(2)], was studied and monitored by LC-MS. The oxidation products identified included products resultant from (a) the insertion of oxygen atoms (1-5), (b) peptide backbone cleavage (short-chain products formed by diamide pathway) and (c) radical-radical crosslinking reactions. In order to identify the modified residues, LC-MS/MS spectra were obtained. The insertion of oxygen atoms into the peptide originated hydroxide, di-hydroxide and/or hydroperoxide derivatives. In addition it was found that the aromatic amino acids are most susceptible to being hydroxylated, while the aliphatic amino acids are more prone to forming hydroperoxides. Oxidation products with double bonds were also identified. The short chain products resulted from the alpha-carbon radical of terminal amino acids (Tyr and Leu). Products resulting from cross-linking reactions between intact carbon-centered peptide radical (with and without one HO group) and a side chain radical (*C(7)H(7)O) were identified. It was found that, although all amino acids residues of the peptide undergo modifications, the N-terminal seems to be prone to oxidative modifications under these conditions.     

Identification of components in waste streams by electrospray and tandem mass spectrometry

Highly polar, non-gas-chromatographable compounds have few unambiguous analysis protocols for environmental applications. A recent environmental investigation, concerning the identification of a non-gas-chromatographable yellow component in chemical waste water and in effluents from a biological wastewater treatment plant required the use of a number of analytical approaches. Electrospray mass spectrometry, tandem mass spectrometry, high-performance liquid chromatography, nuclear magnetic resonance, and molecular spectroscopy of commercial and synthesized chlorodinitrophenol isomers were required in order to identify the specific isomer causing the color. The present report summarizes the electrospray ionization and tandem mass spectrometric studies that were used. The mass spectrometric study shows that two different isomers of chlorodinitrophenol exhibit very different collision-induced dissociation (CID) spectra. Differences in the tandem mass spectra can be attributed to the different structures of the anions formed from these two different isomers. Instrumentation that uses electrospray ionization and produces CID mass spectra and optical absorption spectra in a single analysis may be required in order to produce highly specific information on non-gas-chromatographable compounds found in the environment.     

Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometry

Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (aminooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with liquid chromatography and tandem mass spectrometry has been used. In addition to the target tri-branched conjugate, two other conjugates were characterized allowing documentation of transoximation reactions in peptide chemistry. A fourth conjugate was identified as a side product appearing after the first ligation. Data obtained by low-energy collision-induced dissociation led to a rapid and reliable identification of impurities observed in the (aminooxy)acetyl peptide despite a previous high performance liquid chromatography (HPLC) purification. This highlights the great reactivity of the aminooxy group towards carbonyl-containing compounds.     

Identification of dimethylarsinoyl-riboside derivatives in seaweed by pneumatically assisted electrospray tandem mass spectrometry

Pneumatically-assisted electrospray tandem mass spectrometry (ES MS/MS) was proposed as a technique for the identification of dimethylarsinoyl-riboside derivatives (arsenosugars) in seaweed (Laminaria). Conditions for the acquisition of MS and MS/MS spectra were optimized. A size-exclusion HPLC (SE HPLC) step was developed for the purification of algal extracts prior to ES MS. The arsenosugar fraction was found to elute prior to the majority of other arsenic compounds. The identity of the compounds expected to be arsenosugar compounds was confirmed by the collision induced dissociation (CID) of the relevant protonated molecule ions. An independent confirmation of the identity of analytes was obtained by two-dimensional (size-exclusion–anion exchange) HPLC–ICP MS with signal identification by spiking with the appropriate arsenosugar standards.     

Identification and fragmentation of hydrolyzed aluminum species by electrospray ionization tandem mass spectrometry

Earlier characterization of some hydrolysis products of AlCl₃·6H₂O was confirmed by electrospray ionization tandem mass spectrometry with increasing collision energy of projectile ions. At lower collision energies, the aqua ligands were stripped off. At higher energies, two hydroxo groups formed a bridging oxo group with loss of one water molecule. Aluminum complexes could also capture aqua ligands in the collision chamber so long as the parent ion did not fragment, and the fragment ion spectra broadened toward higher m/z values. The chloro ligands were eliminated as hydrochloric acid. The aluminum cores remained highly intact. Copyright © 2004 John Wiley & Sons, Ltd.     

Identification and assay of underivatized urinary acylcarnitines by paper spray tandem mass spectrometry

A new analytical approach, using paper spray tandem mass spectrometry, has been developed for assay of carnitine and acylcarnitines in urine. Paper spray (PS) is a very promising technique, especially in clinical investigations, because of its simplicity, low cost, and rapid sample preparation. A home-made paper spray device was used for assay of urinary acylcarnitines (C2–C18). The performance of solvents with different elution efficiency and paper substrates with different porosity grade and structure were tested by use of spiked synthetic urine. Tandem mass spectrometry in multiple reaction monitoring (MRM) mode was optimized to obtain better specificity and sensitivity. Analyte signals were evaluated for stability and reproducibility. Calibration with [²H₃]propionylcarnitine (C3-d3), [²H₃]octanoylcarnitine (C8-d3), and [²H₃] palmitoylcarnitine (C16-d3) as internal standards was used for quantification. Very good linearity was obtained, with correlation coefficients >0.99 for C0–C12 and C16 acylcarnitines and >0.96 for C14 and C18 acylcarnitines. Accuracy and precision (RSD, %) of the proposed procedure were tested at concentrations of 0.8, 8, and 20 mg L^?1 with very satisfactory results: overall mean accuracy was 98.9 % and overall mean relative standard deviation 1 %. Limits of detection (LOD) between 6 and 208 μg L^?1 for propionylcarnitine and tetradecanoylcarnitine, respectively, can be regarded as very satisfactory. Application of the method to real urine proved that paper spray tandem mass spectrometry is a simple, rapid, and direct tool (no derivatization is required) for assay of carnitine and C2–C12 acylcarnitines in urine.     

Sequencing membrane proteins by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) is an attractive technique for sequencing membrane proteins because it can be applied to peptides in mixtures that are difficult to separate chromatographically. To evaluate the suitability of MS/MS sequencing for membrane proteins and to develop protocols for the preparation of the cleaved peptides, we employed the well characterized apoproteins of bacteriorhodopsin and bovine rhodopsin, i.e. bacterioopsin and opsin, respectively. Without separation, nine out of ten peptides resulting from cyanogen bromide cleavage of bacterioopsin were detected by fast atom bombardment MS, the single undetected fragment being a tetrapeptide that was presumably hidden in the low-m/z matrix background. Furthermore, MS/MS was used to confirm the sequence of all the peptides detected with m/z values below 3.5 kDa (40% of the protein). Bovine opsin was analyzed in a similar fashion. Tandem MS/MS has thus allowed the sequencing of substantial portions of two integral membrane proteins by the analysis of unseparated peptide mixtures, demonstrating for the first time that this technique can obviate some of the most serious difficulties associated with sequencing membrane proteins, namely the difficult-to-achieve separation of the ‘sticky’ peptide fragments.     

Analysis of oxysterols by electrospray tandem mass spectrometry

Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier, and oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. Oxysterols are difficult to analyze by atmospheric pressure ionization mass spectrometry on account of the absence of basic or acidic functional groups in their structures. In this communication, we report a method for the derivatization and analysis of oxysterols by electrospray mass spectrometry. Oxysterols with a 3beta-hydroxy-Delta5 structure were converted by cholesterol oxidase to 3-oxo-Delta4 steroids and then derivatized with the Girard P reagent to give Girard P hydrazones, which were subsequently analyzed by tandem mass spectrometry. The improvement in sensitivity for the analysis of 25-hydroxycholesterol upon oxidation and derivatization was over 1000.     

Computer-controlled sequencing of peptides by tandem mass spectrometry

A fully automated computer-controlled system was used to generate series of different linked scans at constant B2E and constant neutral loss in the second field-free region. This system has been shown to be suitable for deriving the amino acid sequence of oligopeptides.     

Identification of phosphoramide mustard/DNA adducts using tandem mass spectrometry

The reaction pathway of alkylating agents is often exploited in the design of bifunctional anti-cancer drugs. These drugs form mono-DNA adducts as well as inter- and intra-strand cross-linked adducts, notably by reaction at DNA bases, including the N-7-position of guanine (G). A positive-ion fast-atom bombardment (FAB) mass spectrum of an in vitro preparation of DNA alkylated with phosphoramide mustard (the active metabolite of the anti-cancer drug cyclophosphamide) indicated the presence of the two mono-DNA adducts N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl] amine, designated NOR-G, and N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl] amine, designated NOR-G-OH, (MH+ 257/259 and 239, respectively) but not the presence of the cross-linked adduct N,N-bis-[2-(7-guaninyl)ethyl] amine, designated G-NOR-G (MH+ 372). Using synthetic standards, daughter-ion spectra of NOR-G, NOR-G-OH and G-NOR-G were obtained (matrix 0.2 M p-toluene sulphonic acid in glycerol) by positive-ion FAB tandem mass spectrometry (FAB-MS/MS). The daughter-ion spectra of both mono-DNA adducts NOR-G and NOR-G-OH contained a fragment ion at m/z 152 [G + H]+, whereas the cross-linked adduct, G-NOR-G, showed an ion at m/z 221, [MH-G]+. Evidence for the presence of NOR-G, NOR-G-OH and G-NOR-G in the in vitro preparation was obtained by performing a double parent-ion scan on m/z 152 and 221. The presence of G-NOR-G was further supported by performing a single parent-ion scan on m/z 221.(ABSTRACT TRUNCATED AT 250 WORDS)     

Speciation of organotin compounds by tandem mass spectrometry

The collison-induced dissociation of a range of organotin compounds has been examined by tandem (MS–MS) mass spectrometry in order to investigate the potential of the technique for speciation without prior chromatographic separation. Tables of parent–daughte ion relationships are prepared for a group of six closely related compounds. The results obtained are sufficiently characteristic to distinguish between the isolated compounds and to identify the individual components of a simple mixture. This procedure has potential applications for less volatile compounds and for quantitative analysis.     

Negative ion electrospray and tandem mass spectrometric analysis of platelet activating factor (PAF) (1-hexadecyl-2-acetyl-glycerophosphocholine)

The analysis of 1-hexadecyl-2-acetyl-glycerophosphocholine (platelet activating factor, PAF) by negative ion and normal-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) was investigated as an alternative technique to the currently used gas chromatography/MS and positive ion LC/MS/MS procedures. The positive ion [M + H]+ derived from PAF and generated by electrospray ionization is abundant, but the potential presence of isobaric 1-octadecanoyl-2-lyso-glycerophosphocholine (stearoyl-lyso-GPC) and 1-hexadecanoyl-2-formyl-glycerophosphocholine (PFPC) in biological samples limits the use of the most abundant collision-induced decomposition (CID) transition (formation of the phosphocholine ion, m/z 524-->184) if chromatographic separation is not achieved. Less abundant CID product ions, such as loss of the neutral ketene molecule derived from the respective fatty acyl groups, provide the requisite specificity, but the intensity of these transitions yields a signal-to-noise ratio that greatly diminishes the analytical sensitivity. With negative ion LC/MS/MS, however, the molecular anions [M - 15]- derived from PAF, stearoyl-lyso-GPC and PFPC decompose to the carboxylate anions at m/z 59, 283 and 255, respectively, permitting discrimination of these isobaric molecules even without chromatographic separation. In addition, the CID of [M - 15]- was favorable, yielding ion currents of sufficient intensity to permit the measurement of PAF when isolated from small quantities of biological material. With the use of a stable isotopically labeled variant of PAF and isotope dilution, negative ion LC/MS/MS was found to measure PAF reliably even in the presence of the isobaric stearoyl-lyso-GPC and permitted the use of non-chlorinated mobile phases for normal-phase high-performance LC.     

Pyrrolidine derivatives as inhibitors of platelet aggregation induced by platelet activating factor

Newly synthesized pyrrolidine derivatives inhibited rabbit platelet aggregation induced by platelet activating factor.     

Improved mass accuracy for tandem mass spectrometry

With the emergence of top-down proteomics, the ability to achieve high mass measurement accuracy on tandem MS/MS data will be beneficial for protein identification and characterization. (FT-ICR) Fourier transform ion cyclotron resonance mass spectrometers are the ideal instruments to perform these experiments with their ability to provide high resolution and mass accuracy. A major limitation to mass measurement accuracy in FT-ICR instruments arises from the occurrence of space charge effects. These space charge effects shift the cyclotron frequency of the ions, which compromises the mass measurement accuracy. While several methods have been developed that correct these space charge effects, they have limitations when applied to MS/MS experiments. It has already been shown that additional information inherent in electrospray spectra can be used for improved mass measurement accuracy with the use of a computer algorithm called DeCAL (deconvolution of Coulombic affected linearity). This paper highlights a new application of the strategy for improved mass accuracy in tandem mass analysis. The results show a significant improvement in mass measurement accuracy on complex electron capture dissociation spectra of proteins. We also demonstrate how the improvement in mass accuracy can increase the confidence in protein identification from the fragment masses of proteins acquired in MS/MS experiments.     

Hybrid mass spectrometers for tandem mass spectrometry

Mass spectrometers that use different types of analyzers for the first and second stages of mass analysis in tandem mass spectrometry (MS/MS) experiments are often referred to as "hybrid" mass spectrometers. The general goal in the design of a hybrid instrument is to combine different performance characteristics offered by various types of analyzers into one mass spectrometer. These performance characteristics may include mass resolving power, the ion kinetic energy for collision-induced dissociation, and speed of analysis. This paper provides a review of the development of hybrid instruments over the last 30 years for analytical applications.     

Identification of dimer impurities in ampicillin and amoxicillin by capillary LC and tandem mass spectrometry

A micro-scale liquid chromatography electrospray ionization tandem mass spectrometric method was developed for the identification of polymerized impurities in ampicillin and amoxicillin in aqueous solution. Ampicillin and amoxicillin are broad-spectrum antibiotics and widely used for the treatment of human and animal infections. In this study ampicillin, amoxicillin, and their dimers were trapped in a 5-cm capillary column containing C18 sorbents. The analytes were separated on a reversed-phase column and introduced into the mass spectrometer via a nanospray ion source. An isocratic mobile phase consisting of 1% formic acid-acetonitrile (50:50, v/v) was used. For identification, the fragment ions of the analytes were monitored. The aim of the present study was to develop an optimized quality control method for the analysis of high molecular weight impurities of ampicillin and amoxicillin.     

Identification of factor C protein from Streptomyces griseus by microelectrospray mass spectrometry

Factor C, an extracellular signal protein of cellular differentiation, was studied and significant homology was found to several zinc finger-type regulatory proteins. The complete amino acid sequence, deduced from the gene, that encodes the protein, did not support the hypothesis that this protein might be a zinc finger-type regulatory protein. However, a theoretical single nucleotide insertion in the gene can result in another similarly sized protein containing about 20 His residues, which would be responsible for the high zinc affinity of factor C. The protein sample was reduced, alkylated and then in-gel digested with trypsin. The peptide fragments were then separated by capillary chromatography and identified by microelectrospray mass spectrometry. Peaks of higher intensity were sequenced by tandem mass spectrometry. The identified peptide fragments and the measured molecular mass of factor C protein also confirmed the original sequence of protein, as there was no shift in the open reading frame.