How to discriminate between leucine and isoleucine by low energy ESI-TRAP MS<Superscript>n</Superscript>

In peptide sequencing experiments involving a single step tandem mass acquisition, leucine and isoleucine are indistinguishable because both are characterized by a 113 Da mass difference from the other peptide fragments in the MS2 spectrum. In this work, we propose a new method to distinguish between these two amino acids in consecutive MSn experiments, exploiting a gas-phase fragmentation of isoleucine that leads to a diagnostic 69 Da ion. We used this method to assess the Leu/Ile residues of several synthetic peptides. The procedure was then tested on a tryptic digest of myoglobin, assigning the correct amino acid in the majority of the peptides. This work was performed with an old and low-resolution instrument, thus demonstrating that our method is suitable for a wide number of ion trap mass spectrometers, not necessarily expensive or up-to-date.     

Extending a Tandem Mass Spectral Library to Include MS<Superscript>2</Superscript> Spectra of Fragment Ions Produced In-Source and MS<Superscript>n</Superscript> Spectra

Tandem mass spectral library searching is finding increased use as an effective means of determining chemical identity in mass spectrometry-based omics studies. We previously reported on constructing a tandem mass spectral library that includes spectra for multiple precursor ions for each analyte. Here we report our method for expanding this library to include MS² spectra of fragment ions generated during the ionization process (in-source fragment ions) as well as MS³ and MS⁴ spectra. These can assist the chemical identification process. A simple density-based clustering algorithm was used to cluster all significant precursor ions from MS¹ scans for an analyte acquired during an infusion experiment. The MS² spectra associated with these precursor ions were grouped into the same precursor clusters. Subsequently, a new top-down hierarchical divisive clustering algorithm was developed for clustering the spectra from fragmentation of ions in each precursor cluster, including the MS² spectra of the original precursors and of the in-source fragments as well as the MSⁿ spectra. This algorithm starts with all the spectra of one precursor in one cluster and then separates them into sub-clusters of similar spectra based on the fragment patterns. Herein, we describe the algorithms and spectral evaluation methods for extending the library. The new library features were demonstrated by searching the high resolution spectra of E. coli extracts against the extended library, allowing identification of compounds and their in-source fragment ions in a manner that was not possible before.

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LC-MS<Superscript><Emphasis Type="BoldItalic">n</Emphasis></Superscript> Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS ⁿ . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS ⁿ fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS ⁿ experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS ⁿ methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.     

Structure elucidation of degradation products of the antibiotic amoxicillin with ion trap MS<Superscript>n</Superscript> and accurate mass determination by ESI TOF

Today, it is necessary to identify relevant compounds appearing in discovery and development of new drug substances in the pharmaceutical industry. For that purpose, the measurement of accurate molecular mass and empirical formula calculation is very important for structure elucidation in addition to other available analytical methods. In this work, the identification and confirmation of degradation products in a finished dosage form of the antibiotic drug amoxicillin obtained under stress conditions will be demonstrated. Structure elucidation is performed utilizing liquid chromatography (LC) ion trap MS/MS and MS3 together with accurate mass measurement of the molecular ions and of the collision induced dissociation (CID) fragments by liquid chromatography electro spray ionization time-of-flight mass spectrometry (LC/ESI-TOF).     

Characterization of selected organic compound classes in secondary organic aerosol from biogenic VOCs by HPLC/MS<Superscript><Emphasis Type="Italic">n</Emphasis></Superscript>

The formation of secondary organic aerosols (SOA) has been investigated intensively during the last two decades in numerous field and laboratory studies and a general understanding exists about the major particle-phase products. However, recent studies show that several new product classes, such as esters, peroxides or organosulfates, also have to be considered in order to understand the detailed chemical mechanisms leading to SOA as well as to predict the aerosol mass loadings. For the identification and quantification of these three compound classes as well as for carboxylic SOA compounds, liquid chromatography (LC)/mass spectrometry (MS) is the most appropriate analytical method. In this article we try to summarize briefly the work that has been done for the determination of SOA-related carboxylic acids and we present new LC/tandem MS results on the characterization of esters, peroxides and organosulfates. In contrast to earlier work, the mass-spectrometric characterization of the individual compounds is always based on the comparison with authentic reference compounds.     

New insights in the formation of deoxynucleoside adducts with the heterocyclic aromatic amines PhIP and IQ by means of ion trap MS<Superscript>n</Superscript> and accurate mass measurement of fragment ions

The formation of adducts by reaction of active metabolites of two heterocyclic aromatic amines (NHOH-PhIP and NHOH-IQ) at nucleophilic sites of deoxynucleosides has been studied by LC-MS(n) analyses of the obtained reaction mixtures. Sequential MS(3) experiments were carried out on an ion trap mass spectrometer to gain extensive structural information on each adduct detected in the first MS step. Attribution of ions was supported by accurate mass measurements performed on an Orbitrap mass analyzer. Particular attention was given to ions diagnostic of the linking between the heterocyclic aromatic amine (HAA) and the deoxynucleoside. By this way, the structures of five adducts have been characterized in this study, among which two are new compounds: dG-N7-IQ and dA-N(6)-IQ. No depurinating adduct was found in the reactions investigated therein. As expected, the C8 and N(2) atoms of dG were found as the most reactive sites of deoxynucleosides, resulting in the formation of two different adducts with IQ and one adduct with PhIP. An unusual non-depurinating dG-N7-IQ adduct has been characterized and a mechanism is proposed for its formation on the basis of the reactivity of arylamines. A dA-N(6)-IQ adduct has been identified for the first time in this work, showing that HAAs can generate DNA adducts with bases other than dG.     

Data-dependent neutral gain MS<Superscript>3</Superscript>: Toward automated identification of the N-Oxide functional group in drug metabolites

We report here an automated method for the identification of N-oxide functional groups in drug metabolites by using the combination of liquid chromatography/tandem mass spectrometry (LC/MS ⁿ ) based on ion-molecule reactions and collision-activated dissociation (CAD). Data-dependent acquisition, which has been readily utilized for metabolite characterization using CAD-based methods, is adapted for use with ion-molecule reaction-based tandem mass spectrometry by careful choice of select experimental parameters. Two different experiments utilizing ion-molecule reactions are demonstrated, data-dependent neutral gain MS³ and data-dependent neutral gain pseudo-MS³, both of which generate functional group selective mass spectral data in a single experiment and facilitate increased throughput in structural elucidation of unknown mixture components. Initial results have been generated by using an LC/MS ⁿ method based on ion-molecule reactions developed earlier for the identification of the N-oxide functional group in pharmaceutical samples, a notoriously difficult functional group to identify via CAD alone. Since commercial software and straightforward, external instrument modification are used, these experiments are readily adaptable to the industrial pharmaceutical laboratory.     

Time-space evolution of the cloud of ion-electron pairs formed by Auger electrons around Mossbauer ion <Superscript>57</Superscript>Fe<Superscript><Emphasis Type="Italic">n</Emphasis>+</Superscript>

It is shown that the emission of energetic Auger electrons during formation of the Mossbauer nucleus ⁵⁷Fe leads not only to the formation of multicharged ion ⁵⁷Fe ⁿ⁺, but also to the formation of a cloud of several hundred (200–300) ion-electron pairs (H₂O⁺, e ^?) around the Fe ion. This cloud is called an Auger blob. Its size (radius) is approximately ～100 Å. Fast radiation-chemical reactions in an Auger blob determine the experimentally observable ratio of yields of final chemically stable states (Fe³⁺ and Fe²⁺) of the Mossbauer ion. Knowledge of this ratio is important for an adequate interpretation of the results of Mossbauer emission spectroscopy. Although our assessments relate to iron nuclei in frozen aqueous solutions, they can be easily adjusted for media of other chemical composition.     

Atomic many-body effects in the 4f XPS of the U<Superscript>5+</Superscript> and U<Superscript>4+</Superscript> cations: part II: consequences of orbital relaxation

Ab initio, fully relativistic four component theory was used to determine atomic many-body effects for the 4f X-ray photoelectron spectra (XPS) of U⁵⁺ and U⁴⁺ cations. Many-body effects were included through the use of configuration interaction (CI) wavefunctions, WF‘s, that allow the mixing of XPS allowed and XPS forbidden configurations. This work extends our earlier study of the U 4f XPS in that the orbitals for the final, ionic states of the cations are allowed to relax in the presence of the 4f core-hole. In the earlier work, orbitals optimized for the initial state were frozen and also used for the final, ionic states. While the main XPS features are similar in both cases, using relaxed orbitals for the ionic states introduces changes in the multiplet splitting and in the 4f_5/2 and 4f_7/2 spin–orbit splitting. The extent of configuration mixing for the U⁵⁺ and U⁴⁺ final state WF’s is characterized by the magnitude of the intensity lost by the main peaks to satellites. Overall, the use of relaxed orbitals improves the agreement between the theoretical XPS for the U⁴⁺ cation and the experimental measurements for UO₂.     

Facile one-pot synthesis of nano-zinc hydroxide by electro-dissolution of zinc as a sacrificial anode and the application for adsorption of Th<Superscript>4+</Superscript>, U<Superscript>4+</Superscript>, and Ce<Superscript>4+</Superscript> from aqueous solution

Facilely synthesized zinc hydroxide nanoparticles by electro-dissolution of zinc sacrificial anodes were investigated for the adsorption of thorium (Th⁴⁺), uranium (U⁴⁺) and cerium (Ce⁴⁺) from aqueous solution. Various operating parameters such as effect of pH, current density, temperature, electrode configuration, and electrode spacing on the adsorption efficiency of Th⁴⁺, U⁴⁺ and Ce⁴⁺ were studied. The results showed that the maximum removal efficiency was achieved for Th⁴⁺, U⁴⁺ and Ce⁴⁺ with zinc as anode and stainless steel as cathode at a current density of 0.2 A/dm² and pH of 7.0. First- and second-order rate equations were applied to study the adsorption kinetics. The adsorption process follows second order kinetics model with good correlation. The Langmuir, Freundlich adsorption models were applied to describe the equilibrium isotherms and the isotherm constants were determined. The experimental adsorption data were fitted to the Langmuir adsorption model. Thermodynamic parameters such as free energy (ΔG°), enthalpy (ΔH°), and entropy changes (ΔS°) for the adsorption of Th⁴⁺, U⁴⁺ and Ce⁴⁺ were computed to predict the nature of adsorption process. Temperature studies showed that the adsorption was endothermic and spontaneous in nature.     

An Isotope (<Superscript>18</Superscript>O, <Superscript>15</Superscript>N,and <Superscript>2</Superscript>H) Technique to Investigate the Metal Ion Interactions Between the Phosphoryl Group and Amino Acid Side Chains by Electrospray Ionization Mass Spectrometry

Cationic metal ion-coordinated N-diisopropyloxyphosphoryl dipeptides (DIPP-dipeptides) were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MS ⁿ ). Two novel rearrangement reactions with hydroxyl oxygen or carbonyl oxygen migrations were observed in ESI-MS/MS of the metallic adducts of DIPP-dipeptides, but not for the corresponding protonated DIPP-dipeptides. The possible oxygen migration mechanisms were elucidated through a combination of MS/MS experiments, isotope (¹⁸O, ¹⁵N, and ²H) labeling, accurate mass measurements, and density functional theory (DFT) calculations at the B3LYP/6-31 G(d) level. It was found that lithium and sodium cations catalyze the carbonyl oxygen migration more efficiently than does potassium and participation through a cyclic phosphoryl intermediate. In addition, dipeptides having a C-terminal hydroxyl or aromatic amino acid residue show a more favorable rearrangement through carbonyl oxygen migration, which may be due to metal cation stabilization by the donation of lone pair of the hydroxyl oxygen or aromatic π-electrons of the C-terminal amino acid residue, respectively. It was further shown that the metal ions, namely lithium, sodium, and potassium cations, could play a novel directing role for the migration of hydroxyl or carbonyl oxygen in the gas phase. This discovery suggests that interactions between phosphorylated biomolecules and proteins might involve the assistance of metal ions to coordinate the phosphoryl oxygen and protein side chains to achieve molecular recognition.     

Analysis by Means of <Superscript>1</Superscript>H NMR Spectroscopy of Heteroassociaion in Water Solution of Antitumor Antibiotics Daunomycin and Actinomycin D

Heteroassociation of aromatic antitumor antibiotics daunomycin (DAU) and actinomycin D (AMD) was investigated using 1D and 2D ¹H NMR spectroscopy (at 500 MHz) and molecular mechanics procedure with the goal of establishing the mechanism of the combined action of antibiotics in the system AMD-DAU. The experimental data were processed applying a modified statistical and thermodynamic model of the molecules heteroassociation. Proceeding from this model the values were obtained of induced proton chemical shifts, equilibrium constant and thermodynamic parameters of complexing reaction between DAU and AMD. By means of molecular mechanics with the use of X-PLOR software and of the analysis results of the induced proton chemical shifts in the molecules the most probable spatial structure, 1:1, was established for the heterocomplex of DAU and AMD. Heterocomplexes of daunomycin and actinomycin D form due to stacking interaction between the aromatic chromophores with possible additional stabilization of the complexes by an intermolecular hydrogen bond.     

Quick Determination of the Total Content of Aromatic Hydrocarbons in Reformer Naphthas by <Superscript>1</Superscript>H NMR Spectroscopy

A procedure for fast determination of the total content of arenes in reformer naphthas by ¹H NMR spectroscopy was developed and tested. Their content is determined from the ratio of the integral intensities of the signals of aromatic protons and alkane methyl protons, using a calibration plot constructed on the basis of GLC data.     

Electronic spectra of C<Subscript>6</Subscript>H<Superscript>+</Superscript> and C<Subscript>6</Subscript>H<Stack><Subscript>3</Subscript><Superscript>+</Superscript></Stack> in the gas phase

Measurement of the ³Π-³Π transition of C₆H⁺ in the gas phase near 19486 cm⁻¹ is reported. The experiment was carried out with a supersonic slit-jet expansion discharge using cavity ringdown absorption spectroscopy. Partly resolved P lines and observation of band heads permitted a rotational contour fit. Spectroscopic constants in the ground and excited-state were determined. The density of ions being sampled is merely 2×10⁸ cm⁻³. Broadening of the spectral lines indicates the excited-state lifetime to be ≈100 ps. The electronic transition of HC₆H₂⁺ at 26402 cm⁻¹ assumed to be ¹A₁-X¹A₁ in C_2v symmetry could not be rotationally resolved.     

Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by <Superscript>13</Superscript>C-/<Superscript>12</Superscript>C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by ¹³C-dansyl and ¹²C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.     

Hydration of the H<Superscript>+</Superscript>, Li<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, and Cs<Superscript>+</Superscript> ions in MF-4SK perfluorinated sulfonic acid cation-exchange membranes modified with inorganic dopants

The hydration, state, and mobility of protons and Li⁺, Na⁺, and Cs⁺ ions in MF-4SK perfluorinated sulfonic acid cation-exchange membranes doped with silicon dioxide and phosphotungstic acid have been investigated by NMR and impedance spectroscopy. The dopants increase the moisture content of the membrane and change the system of pores and channels in which ion transport takes place. At low humidities, the dopant particles are involved in ion transport. The greatest effect is observed for the membranes doped with both SiO₂ and phosphotungstic acid. The water molecules sorbed by dopant particles as a material participate in the hydration of alkali metal cations in the membrane.     

Discrimination of GalNAc (4S/6S) sulfation sites in chondroitin sulfate disaccharides by chip-based nanoelectrospray multistage mass spectrometry

Sulfation pattern within chondroitin sulfate (CS) glycosaminoglycan (GAG) chains is an important post-translational modification that regulates their interaction with proteins. In this context, development of highly efficient and reproducible analytical methods for the investigation of CS sulfation patterns is of high necessity. In this study we report a novel method for straightforward determination of N-acetylgalactosamine (GalNAc) sulfation sites in chondroitin sulfate disaccharides. Our protocol involves combining fully automated chip-based nanoelectrospray (nanoESI) for analyte infusion and ionization in negative ion mode with multistage (MSⁿ) collision-induced dissociation (CID) high capacity ion trap (HCT) mass spectrometry for generation of sequence ions diagnostic for identification of sulfate ester group position within GalNAc residues. The feasibility of this approach is here demonstrated on chondroitin 6-O-sulfate and chondroitin 4-O-sulfate disaccharides. Fragmentation patterns obtained by MS² and MS³ sequencing stages provided first mass spectrometric data from which sulfation site(s) within GalNAc monosaccharide ring could be unequivocally deciphered. Hence, the method allowed discriminating 4S/6S sulfation sites solely on the basis of MS and multistage MS evidence.      

Photochemical substitution of benzene in the iron cyclohexadienyl complex [(η<Superscript>5</Superscript>-C<Subscript>6</Subscript>H<Subscript>7</Subscript>)Fe(η-C<Subscript>6</Subscript>H<Subscript>6</Subscript>)]<Superscript>+</Superscript>

The visible light irradiation of the [(η⁵-C₆H₇)Fe(η-C₆H₆)]⁺ cation (1) in acetonitrile resulted in the substitution of the benzene ligand to form the labile acetonitrile species [(η⁵-C₆H₇)Fe(MeCN)₃]⁺ (2). The reaction of 1 with Bu^tNC in MeCN produced the stable isonitrile complex [(η⁵-C₆H₇)Fe(Bu^tNC)₃]⁺ (3). The photochemical reaction of cation 1 with pentaphosphaferrocene Cp*Fe(η-cyclo-P₅) afforded the triple-decker cation with the bridging pentaphospholyl ligand, [(η⁵-C₆H₇)Fe(μ-η:η-cyclo-P₅)FeCp*]⁺ (4). The latter complex was also synthesized by the reaction of cation 2 with Cp*Fe(η-cyclo-P₅). The structure of the complex [3]PF₆ was established by X-ray diffraction. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2088–2091, November, 2007.     

Analysis of <Superscript>26</Superscript>Al in meteorite samples by coincidence gamma-ray spectrometry

Large volume Ge-detectors have been used for the analysis of positron emitters of cosmogenic origin in extraterrestrial samples including lunar samples and meteorites. We present results from the analysis of ²⁶Al in the Rumanová chondrite which was found in 1994 in Slovakia. A slide of the meteorite was cut into ~1 cm³ cubes which were analyzed in a coincidence Ge–NaI(Tl) spectrometer placed in a large shield measuring 1.5 × 1.5 × 2 m³, and consisting of iron, lead and copper layers. Operational characteristics of the spectrometer are presented and discussed, as well as the ²⁶Al profile observed in the meteorite.