bluuebunny/crossref_metadata_2025 huggingface dataset:
Why are proteins marginally stable?
Authors: Taverna Darin M. , Goldstein Richard A.
Published: September 2002

Most globular proteins are marginally stable regardless of size or activity. The most common interpretation is that proteins must be marginally stable in order to function, and so marginal stability represents the results of positive selection. We consider the issue of marginal stability directly using model proteins and the dynamical aspects of protein evolution in populations. We find that the marginal stability of proteins is an inherent property of proteins due to the high dimensionality of the sequence space, without regard to protein function. In this way, marginal stability can result from neutral, non‐adaptive evolution. By allowing evolving protein sub‐populations with different stability requirements for functionality to complete, we find that marginally stable populations of proteins tend to dominate. Our results show that functionalities consistent with marginal stability have a strong evolutionary advantage, and might arise because of the natural tendency of proteins towards marginal stability. Proteins 2002;46:105–109. © 2001 Wiley‐Liss, Inc.
Intrinsic structural disorder and sequence features of the cell cycle inhibitor p57Kip2
Authors: Adkins Joshua N. , Lumb Kevin J.
Published: September 2002

The cell cycle inhibitor p57Kip2 induces cell cycle arrest by inhibiting the activity of cyclin‐dependent kinases. p57, although active as a cyclin A‐CDK2 inhibitor, is largely unfolded or intrinsically disordered as shown by circular dichroism and fluorescence spectra characteristic of an unfolded protein and a hydrodynamic radius consistent with an unfolded structure. In addition, the N‐terminal domain of p57 is both functionally independent as a cyclin A‐CDK2 inhibitor and unstructured, as demonstrated by circular dichroism and fluorescence spectra indicative of unfolded proteins, a lack of 1H chemical shift dispersion and a hydrodynamic radius consistent with a highly unfolded structure. The amino acid compositions of full‐length p57 and the excised QT domain of p57 exhibit significant deviations from the average composition of globular proteins that are consistent with the observed intrinsic disorder. However, the amino acid composition of the CDK inhibition domain of p57 does not exhibit such a striking deviation from the average values observed for proteins, implying that a general low level of hydrophobicity, rather than depletion or enrichment in specific amino acids, contributes to the intrinsic disorder of the excised p57 CDK inhibition domain. Proteins 2002;46:1–7. © 2001 Wiley‐Liss, Inc.
Exploratory studies of ab initio protein structure prediction: Multiple copy simulated annealing, AMBER energy functions, and a generalized born/solvent accessibility solvation model
Authors: Liu Yongxing , Beveridge D. L.
Published: September 2002

A theoretical and computational approach to ab initio structure prediction for polypeptides in water is described and applied to selected amino acid sequences for testing and preliminary validation. The method builds systematically on the extensive efforts applied to parameterization of molecular dynamics (MD) force fields, employs an empirically well‐validated continuum dielectric model for solvation, and an eminently parallelizable approach to conformational search. The effective free energy of polypeptide chains is estimated from AMBER united atom potential functions, with internal degrees of freedom for both backbone and amino acid side chains explicitly treated. The hydration free energy of each structure is determined using the Generalized Born/Solvent Accessibility (GBSA) method, modified and reparameterized to include atom types consistent with the AMBER force field. The conformational search procedure employs a multiple copy, Monte Carlo simulated annealing (MCSA) protocol in full torsion angle space, applied iteratively on sets of structures of progressively lower free energy until a prediction of a structure with lowest effective free energy is obtained. Calibration tests for the effective energy function and search algorithm are performed on the alanine dipeptide, selected protein crystal structures, and united atom decoys on barnase, crambin, and six examples from the Rosetta set. Specific demonstration cases of the method are provided for the 8‐mer sequence of Ala residues, a 12‐residue peptide with longer side chains QLLKKLLQQLKQ, a de novo designed 16 residue peptide of sequence (AAQAA)3Y, a 15‐residue sequence with a β sheet motif, GEWTWDATKTFTVTE, and a 36 residue small protein, Villin headpiece. The Ala 8‐mer readily formed an α‐helix. An α‐helix structure was predicted for the 16‐mer, consistent with observed results from IR and CD spectroscopy and with the pattern in ψ/ϕ angles of known protein structures. The predicted structure for the 12‐mer, composed of a mix of helix and less regular elements of secondary structure, lies 2.65 Å RMS from the observed crystal structure. Structure prediction for the 8‐mer β‐motif resulted in form 4.50 Å RMS from the crystal geometry. For Villin, the predicted native form is very close to the crystal structure, RMS values of 3.5 Å (including sidechains), and 1.01 Å (main chain only). The methodology permits a detailed analysis of the molecular forces which dominate various segments of the predicted folding trajectory. Analysis of the results in terms of internal torsional, electrostatic and van der Waals and the electrostatic and non‐electrostatic contributions to hydration, including the hydrophobic effect, is presented. Proteins 2002;46:128–146. © 2001 Wiley‐Liss, Inc.
Calculation of protein ionization equilibria with conformational sampling: pKa of a model leucine zipper, GCN4 and barnase
Authors: Gorfe Alemayehu A. , Ferrara Philippe , Caflisch Amedeo , Marti Daniel N. , Bosshard Hans Rudolf , Jelesarov Ilian
Published: September 2002

The use of conformational ensembles provided by nuclear magnetic resonance (NMR) experiments or generated by molecular dynamics (MD) simulations has been regarded as a useful approach to account for protein motions in the context of pKa calculations, yet the idea has been tested occasionally. This is the first report of systematic comparison of pKa estimates computed from long multiple MD simulations and NMR ensembles. As model systems, a synthetic leucine zipper, the naturally occurring coiled coil GCN4, and barnase were used. A variety of conformational averaging and titration curve‐averaging techniques, or combination thereof, was adopted and/or modified to investigate the effect of extensive global conformational sampling on the accuracy of pKa calculations. Clustering of coordinates is proposed as an approach to reduce the vast diversity of MD ensembles to a few structures representative of the average electrostatic properties of the system in solution. Remarkable improvement of the accuracy of pKa predictions was achieved by the use of multiple MD simulations. By using multiple trajectories the absolute error in pKa predictions for the model leucine zipper was reduced to as low as approximately 0.25 pKa units. The validity, advantages, and limitations of explicit conformational sampling by MD, compared with the use of an average structure and a high internal protein dielectric value as means to improve the accuracy of pKa calculations, are discussed. Proteins 2002;46:41–60. © 2001 Wiley‐Liss, Inc.
Automated docking to multiple target structures: Incorporation of protein mobility and structural water heterogeneity in AutoDock
Authors: Österberg Fredrik , Morris Garrett M. , Sanner Michel F. , Olson Arthur J. , Goodsell David S.
Published: September 2002

Protein motion and heterogeneity of structural waters are approximated in ligand‐docking simulations, using an ensemble of protein structures. Four methods of combining multiple target structures within a single grid‐based lookup table of interaction energies are tested. The method is evaluated using complexes of 21 peptidomimetic inhibitors with human immunodeficiency virus type 1 (HIV‐1) protease. Several of these structures show motion of an arginine residue, which is essential for binding of large inhibitors. A structural water is also present in 20 of the structures, but it must be absent in the remaining one for proper binding. Mean and minimum methods perform poorly, but two weighted average methods permit consistent and accurate ligand docking, using a single grid representation of the target protein structures. Proteins 2002;46:34–40. © 2001 Wiley‐Liss, Inc.
Reversible substrate‐induced domain motions in ribonuclease A
Authors: Vitagliano Luigi , Merlino Antonello , Zagari Adriana , Mazzarella Lelio
Published: September 2002

Despite the increasing number of successful determinations of complex protein structures the understanding of their dynamics properties is still rather limited. Using X‐ray crystallography, we demonstrate that ribonuclease A (RNase A) undergoes significant domain motions upon ligand binding. In particular, when cytidine 2′‐monophosphate binds to RNase A, the structure of the enzyme becomes more compact. Interestingly, our data also show that these structural alterations are fully reversible in the crystal state. These findings provide structural bases for the dynamic behavior of RNase A in the binding of the substrate shown by Petsko and coworkers (Rasmussen et al. Nature 1992;357:423–424). These subtle domain motions may assume functional relevance for more complex system and may play a significant role in the cooperativity of oligomeric enzymes. Proteins 2002;46:97–104. © 2001 Wiley‐Liss, Inc.
Continuum electrostatic analysis of irregular ionization and proton allocation in proteins
Authors: Koumanov Assen , Rüterjans Heinz , Karshikoff Andrey
Published: September 2002

Irregular (nonsigmoidal) ionization behavior of titratable groups in proteins is analyzed theoretically, using a computational algorithm designed to count explicitly for tautomers of titratable groups and different locations of polar hydrogens. On the basis of calculations for different model systems (acid–acid, base–base, acid–base pairs, and cluster of three strongly interacting groups), it is demonstrated that the pKvalues, extracted from nonsigmoidal titration curves by fitting to a sum of Henderson–Hasselbalch equations, do not describe the ionization equilibrium correctly. The conditions for observation of irregular titration curves are derived analytically for the case of arbitrary couple of interacting ionizable groups. A possible relation between irregularly shaped titration curves and tautomerization is also illustrated. The protonation–deprotonation equilibrium of Asp76 in ribonucleaseT1is shown to be coupled to dipole reorientation of a water molecule bound at the protein–solvent interface. This finding provides a new interpretation of the experimentally observed chemical shift of this residue. Proteins 2002;46:85–96. © 2001 Wiley‐Liss, Inc.
What are the dielectric “constants” of proteins and how to validate electrostatic models?
Authors: Schutz Claudia N. , Warshel Arieh
Published: September 2002

Implicit models for evaluation of electrostatic energies in proteins include dielectric constants that represent effect of the protein environment. Unfortunately, the results obtained by such models are very sensitive to the value used for the dielectric constant. Furthermore, the factors that determine the optimal value of these constants are far from being obvious. This review considers the meaning of the protein dielectric constants and the ways to determine their optimal values. It is pointed out that typical benchmarks for validation of electrostatic models cannot discriminate between consistent and inconsistent models. In particular, the observed pKa values of surface groups can be reproduced correctly by models with entirely incorrect physical features. Thus, we introduce a discriminative benchmark that only includes residues whose pKa values are shifted significantly from their values in water. We also use the semimacroscopic version of the protein dipole Langevin dipole (PDLD/S) formulation to generate a series of models that move gradually from microscopic to fully macroscopic models. These include the linear response version of the PDLD/S models, Poisson Boltzmann (PB)‐type models, and Tanford Kirkwwod (TK)‐type models. Using our different models and the discriminative benchmark, we show that the protein dielectric constant, εp, is not a universal constant but simply a parameter that depends on the model used. It is also shown in agreement with our previous works that εp represents the factors that are not considered explicitly. The use of a discriminative benchmark appears to help not only in identifying nonphysical models but also in analyzing effects that are not reproduced in an accurate way by consistent models. These include the effect of water penetration and the effect of the protein reorganization. Finally, we show that the optimal dielectric constant for self‐energies is not the optimal constant for charge‐charge interactions. Proteins 2001;44:400–417. © 2001 Wiley‐Liss, Inc.
Modeling of the metallo‐β‐lactamase from B. fragilis: Structural and dynamic effects of inhibitor binding
Authors: Salsbury Freddie R. , Crowley Michael F. , Brooks Charles L.
Published: September 2002

The structure and dynamics of an inhibitor‐bound complex of the metallo‐β‐lactamase from Bacteroides fragilis are studied by using molecular dynamics. A search of the conformational space was performed to obtain three distinct models of the complex, which were then subjected to solvated molecular dynamics. A solvated molecular dynamics study of the apo protein was performed to serve as a baseline for comparison with the bound simulations. We find loop conformation changes due to binding as well as a decrease in flexibility of the protein as a whole and especially in the major loop of the β‐lactamase. We report the structural and dynamical features of the inhibitor‐bound and apo models, as well as experimentally measurable quantities, which should be capable of distinguishing the two binding modes we have determined. Proteins 2001;44:448–459. © 2001 Wiley‐Liss, Inc.
Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand‐binding core in the presence of glutamate and kainate
Authors: Mendieta Jesús , Ramírez Galo , Gago Federico
Published: September 2002

Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs) through the induced transient opening of transmembrane ion channels. The three‐dimensional structure of the extracellular ligand‐binding core of iGluRs shares the overall features of bacterial periplasmic binding proteins (PBPs). In both families of proteins, the ligand‐binding site is arranged in two domains separated by a cleft and connected by two peptide stretches. PBPs undergo a typical hinge motion of the two domains associated with ligand binding that leads to a conformational change from an open to a closed form. The common architecture suggests a similar closing mechanism in the ligand‐binding core of iGluRs induced by the binding of specific agonists. Starting from the experimentally determined kainate‐bound closed form of the S1S2 GluR2 construct, we have studied by means of molecular dynamics simulations the opening motion of the ligand‐binding core in the presence and in the absence of both glutamate and kainate. Our results suggest that the opening/closing interdomain hinge motions are coupled to conformational changes in the insertion region of the transmembrane segments. These changes are triggered by the interaction of the agonists with the essential Glu 209 residue. A plausible mechanism for the coupling of agonist binding to channel gating is discussed. Proteins 2001;44:460–469. © 2001 Wiley‐Liss, Inc.
Structure of a legume lectin from the bark of Robinia pseudoacacia and its complex with N‐acetylgalactosamine
Authors: Rabijns A. , Verboven C. , Rougé P. , Barre A. , Van Damme E.J.M. , Peumans W.J. , De Ranter C.J.
Published: September 2002

The structure of the bark lectin RPbAI (isoform A4) from Robinia pseudoacacia has been determined by protein crystallography both in the free form and complexed with N‐acetylgalactosamine. The free form is refined at 1.80 Å resolution to an R‐factor of 18.9% whereas the complexed structure has an R‐factor of 19.7% at 2.05 Å resolution. Both structures are compared to each other and to other available legume lectin structures. The polypeptide chains of the two structures exhibit the characteristic legume lectin tertiary fold. The quaternary structure resembles that of the Phaseolus vulgaris lectin, the soybean agglutinin, and the Dolichos biflorus lectin, but displays some unique features leading to the extreme stability of this lectin. Proteins 2001;44:470–478. © 2001 Wiley‐Liss, Inc.
Studies on a new group of biodegradable surfactants for glyphosate
Authors: Haefs Roland , Schmitz‐Eiberger Michaela , Mainx Hans‐G , Mittelstaedt Werner , Noga Georg
Published: September 2002

The effectiveness of a homologous series of biodegradable rapeseed oil derivatives (triglyceride ethoxylates; Agnique RSO® series containing an average of 5, 10, 30 and 60 units of ethylene oxide (EO) as adjuvants for foliage‐applied, water‐soluble, systemic active ingredients was evaluated employing glyphosate as an example. Previous experiments had revealed that the surfactants used are not phytotoxic at concentrations ranging from 1 to 10 g litre−1. The experiments were performed usingPhaseolus vulgarisL and nine selected weed species, grown in a growth chamber at 25/20 (± 2) °C day/night temperature and 40/70 (±10)% relative humidity. The surfactants were evaluated for enhancement of spray retention, and foliar penetration biological efficacy of glyphosate. Glyphosate was applied at a concentration of 43 mM. The surfactants were added at concentrations of 1 g litre−1. The commercial glyphosate 360 g AE litre−1SL Roundup Ultra and unformulated glyphosate served as references. The surfactants used improved spray retention, foliar penetration and biological efficacy. Some of the formulations were comparable to the performance of Roundup Ultra in the aspects evaluated; some were even more effective in enhancing spray liquid retention and promoting glyphosate phytotoxicity in several plant species. In these studies Agnique RSO 60 generally was most effective.

© 2002 Society of Chemical Industry
Quantitative screening and matrix effect studies of drug discovery compounds in monkey plasma using fast‐gradient liquid chromatography/tandem mass spectrometry
Authors: Hsieh Yunsheng , Chintala Madhu , Mei Hong , Agans Jacqueline , Brisson Jean‐Marc , Ng Kwokei , Korfmacher Walter A.
Published: September 2002

A higher‐throughput bioanalytical method based on fast‐gradient (1 min run time) high‐performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) was developed for screen‐type analyses of plasma samples from early drug discovery studies in support of exploratory pharmacodynamic studies. The HPLC system equipped with minibore column was interfaced with either atmospheric pressure chemical ionization (APCI) or electrospray (ESI) ionization techniques. The matrix ion suppression effect of both quantitative HPLC/MS/MS analyses was compared using the post‐column infusion system. The use of the described methods provided advantages such as a shorter chromatographic region of ion suppression, less solvent consumption and shorter run times in comparison with standard analytical column HPLC/MS/MS methods. The analytical results obtained by both HPLC/MS/MS methods were in good agreement (within 15% of error) and displayed a good correlation with the pharmacodynamic outcome. Copyright © 2001 John Wiley & Sons, Ltd.
Electrospray tandem mass spectrometry of aminochromes
Authors: Lemos‐Amado Francisco , Domingues Pedro , Ferrer‐Correia A. , Remião Fernando , Milhazes Nuno , Borges Fernanda , Carvalho Félix D. , Bastos Maria L.
Published: September 2002

The catecholamines adrenaline, noradrenaline, dopamine, dopa and isoprenaline were oxidized into their respective aminochromes: adrenochrome, noradrenochrome, dopaminochrome, dopachrome and isoprenochrome. Tandem mass spectrometry (MS/MS) fragmentation patterns were examined for the five aminochromes in order to establish a general structural assignment of these oxidation products by electrospray mass spectrometry. Although protonated aminochromes undergo similar fragmentation patterns with a characteristic consecutive loss of two carbonyl groups, the presence of different substituents in the parent compounds led to significant changes in the CID spectra. This feature is more evident for isoprenochrome and dopachrome, especially for the latter where the MS/MS spectrum is dominated by the loss of formic acid. A general pattern of fragmentation for aminochromes is proposed, which should provide a suitable basis to aid their characterization in studies in vivo or in vitro. Copyright © 2001 John Wiley & Sons, Ltd.
Direct characterization of iodine covalently bound to fulvic acids by electrospray mass spectrometry
Authors: Moulin Valérie , Reiller Pascal , Amekraz Badia , Moulin Christophe
Published: September 2002

Numerous studies have demonstrated that humic substances (HS) react with iodine to form iodo derivatives and thereby can control the bioavailability of radioisotopes. Unfortunately, none of these studies have provided detailed insights into product compounds and so far, to our knowledge, the direct analysis of these species by electrospray ionization (ESI) mass spectrometry has not been explored. The reactivity of iodine with fulvic acids (FA) present in HS was investigated by means of ESI coupled with a quadrupole time‐of‐flight (Q‐TOF) mass spectrometer. ESI spectra of solutions, that were indicated by MS/MS analysis to have formed iodinated species, apparently displayed singly charged ions corresponding in m/z to hypothesized species, viz., [RI − H]−, R = (substituted FA compound). MS/MS analysis based on the diagnostic fragment ions for FA compounds and their iodo derivatives suggests that FA undergo aromatic substitutions. Furthermore, significant differences in mass profiles are observed that presumably result from extended redox reactions. The ESI‐MS technique opens up new opportunities to understand the scavenging properties of HS towards radionuclides and heavy metals for environmental studies. Copyright © 2001 John Wiley & Sons, Ltd.
Determination of anabolic steroids with gas chromatography–ion trap mass spectrometry using hydrogen as carrier gas
Authors: Impens Sandra , Wasch Katia De , Brabander Hubert De
Published: September 2002

Helium is considered to be the ideal carrier gas for gas chromatography/mass spectrometry (GC/MS) in general, and for use with an ion trap in particular. Helium is an inert gas, can be used without special precautions for security and, moreover, it is needed as a damping gas in the trap. A disadvantage of helium is the high viscosity resulting in long GC run times. In this work hydrogen was tested as an alternative carrier gas for GC in performing GC/MS analyses. A hydrogen generator was used as a safe source of hydrogen gas. It is demonstrated that hydrogen can be used as a carrier gas for the gas chromatograph in combination with helium as make‐up gas for the trap. The analysis time was thus shortened and the chromatographic performance was optimized. Although hydrogen has proven useful as a carrier gas in gas chromatography coupled to standard detectors such as ECD or FID, its use is not mentioned extensively in the literature concerning gas chromatography–ion trap mass spectrometry. However, it is worth considering as a possibility because of its chromatographic advantages and its advantageous price when using a hydrogen generator. Copyright © 2001 John Wiley & Sons, Ltd.
Determination of benzene at trace levels in air by a novel method based on solid‐phase microextraction gas chromatography/mass spectrometry
Authors: Saba Alessandro , Cuzzola Angela , Raffaelli Andrea , Pucci Sergio , Salvadori Piero
Published: September 2002

A new method for the determination of benzene at trace levels in air is presented. The method consists of the collection of air samples on adsorbent cartridges with simultaneous adsorption of pre‐established amounts of D6‐labeled internal standard. Desorption from the cartridge is performed by solid‐phase microextraction (SPME) with analysis by gas chromatography/mass spectrometry (GC/MS) using an ion trap mass spectrometer. The influence of several parameters (type of SPME fiber, temperature, time, for example) was investigated, and good linearity in the range 10–400 ng of C6D6, with a coefficient of variance (CV) around 3–5%, was obtained. The method was tested by sampling air in a town center in Italy, and a benzene concentration of approximately 50 µg/m3 was determined. The maximum limit recommended by the European Community is 10 µg/m3. Copyright © 2001 John Wiley & Sons, Ltd.
Automated molecular weight assignment of electrospray ionization mass spectra
Authors: Williams Jon D. , Weiner Brian E. , Ormand James R. , Brunner Jimmy , Thornquest Alan D. , Burinsky David J.
Published: September 2002

Process improvements in the synthesis of therapeutic agents and their intermediates are often facilitated by identification of reaction by‐products. Analysis by liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization is a powerful approach for obtaining molecular weight information for these compounds. Such analyses are well suited for ‘open‐access’ mass spectrometry using generic chromatographic conditions, provided spectral interpretation for unknown compounds is facile. We have developed a software application (MassAssign) that facilitates automated data processing and molecular weight assignment for chromatographic peaks detected by any standard ultraviolet‐visible wavelength detector. The program assigns [M + H]+ ions (and thus molecular weight) in the mass spectra using predetermined criteria. This evaluation process differentiates [M + H]+ ions from other signals in a complex mass spectrum such as those resulting from chromatographic coelution or the presence of multiple species (i.e., fragment ions, singly charged ions, doubly charged ions, adduct ions, proton‐bound dimers, etc.). Once the program has evaluated all ions in a mass spectrum that exceed a preset abundance threshold, MassAssign reports either a numeric value—indicating the chromatographic peak consists of a single component having the displayed molecular weight, ‘MC’—indicating the peak consisted of multiple components, or ‘ND’—that a molecular weight could not be determined unequivocally. The performance of the program was evaluated by comparing mass assignments made by MassAssign against manual interpretation for 55 samples analyzed by positive electrospray ionization using a generic HPLC method. Correct molecular weight assignments were obtained in 90% of the cases. Copyright © 2001 John Wiley & Sons, Ltd.
Collision‐induced dissociation of glycero phospholipids using electrospray ion‐trap mass spectrometry
Authors: Larsen Åsmund , Uran Steinar , Jacobsen Petter Balke , Skotland Tore
Published: September 2002

Characterisation of phospholipids was achieved using collision‐induced dissociation (CID) with an ion‐trap mass spectrometer. The product ions were compared with those obtained with a triple quadrupole mass spectrometer. In the negative ion mode the product ions were mainly sn‐1 and sn‐2 lyso‐phospholipids with neutral loss of ketene in combination with neutral loss of the polar head group. Less abundant product ions were sn‐1 and sn‐2 carboxylate anions. CID using a triple quadrupole mass spectrometer, however, gave primarily the sn‐1 and sn‐2 carboxylate anions together with lyso‐phosphatidic acid with neutral loss of water. For the ion trap a charge‐remote‐type mechanism is proposed for formation of the lyso‐phospholipid product ions by loss of α‐hydrogen on the fatty acid moiety, electron rearrangement and neutral loss of ketene. A second mechanism involves nucleophilic attack of the phosphate oxygen on the sn‐1 and sn‐2 glycerol backbone to form carboxylate anions with neutral loss of cyclo lyso‐phospholipids. CID (MS3 and MS4) of the lyso‐phospholipids using the ion‐trap gave the same carboxylate anions as those obtained with a triple quadrupole instrument where multiple collisions in the collision cell are expected to occur. The data demonstrate that phospholipid species determination can be performed by using LC/MSn with an ion‐trap mass spectrometer with detection of the lyso‐phospholipid anions. The ion‐trap showed no loss in sensitivity in full scan MSn compared to multiple reaction monitoring data acquisition. In combination with on‐line liquid chromatography this feature makes the ion‐trap useful in the scanning modes for rapid screening of low concentrations of phospholipid species in biological samples as recently described (Uran S, Larsen Å, Jacobsen PB, Skotland T. J. Chromatogr. B 2001; 758: 265). Copyright © 2001 John Wiley & Sons, Ltd.
Effect of impurities on the matrix‐assisted laser desorption/ionization mass spectra of insulin
Authors: Bajuk Anna , Głuch Krzysztof , Michalak Leszek
Published: September 2002

The effect of impurity (sodium sulfate Na2SO4; copper sulfate CuSO4; potassium ferrocyanide K4Fe(CN)6; and triammonium citrate (NH4)3C6H5O7) concentration on the positive matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) spectra of insulin is described. 2,5‐Dihydroxybenzoic acid was used as a desorption matrix. The estimated maximum concentrations of tested salts for which no insulin signal was observed were determined. Copyright © 2001 John Wiley & Sons, Ltd.
RSR13, a potential athletic performance enhancement agent: detection in urine by gas chromatography/mass spectrometry
Authors: Breidbach Andreas , Catlin Don H.
Published: September 2002

RSR13 (2‐[4‐[[(3,5‐dimethylanilino)carbonyl]methyl]phenoxyl]‐2‐methylpropionic acid) is a synthetic allosteric modifier of hemoglobin that is currently in a phase III clinical trial as a radio‐enhancing agent. RSR13 has been shown to increase maximum oxygen uptake (VO2max) in a canine skeletal model, which makes it a potential performance‐enhancing agent for endurance athletes, since VO2max is an index of aerobic capacity. In this study we present a method for the detection of RSR13‐bis‐TMS in human urine by gas chromatography/electron impact ionization mass spectrometry (GC/EI‐MS) suitable for doping control laboratories. The presence of RSR13 is detected by monitoring the ions m/z 485 ([M]+·) and 470 ([M − CH3]+). The limit of detection (LOD) is less than 2 ng/mL in urine. Urine samples collected from clinical trial subjects immediately prior to receiving an infusion of RSR13 showed no evidence of RSR13, whereas post‐infusion urine samples contained up to 1181 µg/mL. A urine sample collected 36 h after administration of a small dose (10 mg/kg) and diluted 100‐fold showed a signal 80 times higher than the LOD. Urine samples obtained from 100 randomly selected athletes in our routine testing program did not show any traces of RSR13. Sport authorities may wish to add RSR13 to the list of prohibited substances. Copyright © 2001 John Wiley & Sons, Ltd.
Positive ion electrospray ionization mass spectrometry of double‐stranded DNA/drug complexes
Authors: Gupta Rajesh , Kapur Amit , Beck Jennifer L. , Sheil Margaret M.
Published: September 2002

An Erratum has been published for this article in Rapid Communicatons in Mass Spectrometry 16(7) 2002,740–741.

Positive ion electrospray ionization mass spectra of 16 base‐pair double‐stranded (ds)DNA have been obtained with essentially no ions from single‐stranded DNA present. Single‐stranded DNA was minimized by: (1) careful choice of DNA sequences; (2) the use of a relatively high salt concentration (0.1 M ammonium acetate, pH 8.5), and, (3) a low desolvation temperature (40 °C). Similarly, ESI‐MS complexes of dsDNA with cisplatin, daunomycin and distamycin were obtained that contained only negligible amounts of single‐stranded DNA. The complexes with daunomycin and distamycin were more stable to strand separation in the gas phase than dsDNA alone. This is in agreement with solution studies and with other recent gas phase results. These data contrast with many earlier ESI‐MS studies of dsDNA and DNA/drug complexes in which ions from ssDNA are also normally observed. Copyright © 2001 John Wiley & Sons, Ltd.
18O Labeling: a tool for proteomics
Authors: Stewart Ian I , Thomson Ty , Figeys Daniel
Published: September 2002

An evaluation of the proteolytic labeling and quantification of proteins for diagnostic purposes using trypsin and 18O‐enriched H2O is presented. We demonstrate that comparative or relative quantitation can be performed effectively with this approach. We have developed a protocol that allows the conservation of the labeled peptides in natural abundance water without fear of back‐exchange providing that pH is sufficiently low to quench the catalytic activity of trypsin, but not so low as to promote chemical back‐exchange. Because the labeling efficiency depends on the nature of the peptide, a simple linear relationship between the relative 16O/18O digest buffer mixture content (x) and labeling efficiency (y) does not exist; rather it follows a probability based y = x2 relationship. As such, the extent of peptide labeling using 16O/18O digest buffer mixture ratios may deviate significantly from that expected based on a linear relationship. The evaluation of the relative Ziptip efficiency indicated a loss in sample recovery as the peptide concentration was reduced using normal conditions, suggesting that there is a limit below which there are diminishing returns. In addition, the adsorptive losses due to Speedvac dry down and recovery indicated modest (20%) losses that may vary widely (0–50%) from peptide to peptide. The in‐solution digestion efficiency of standard protein mixtures as a function of concentration revealed a linear decrease with decreasing concentration. This is consistent with enzyme kinetic effects and emphasizes a potential quantitation error that could arise when evaluating differential expression based on peptide detection. The results from our studies demonstrate the power of 18O labeling as an optimization tool for proteomics process development. Copyright © 2001 John Wiley & Sons, Ltd.
Electrospray mass spectrometry of hydrophobic compounds using dimethyl sulfoxide and dimethylformamide as solvents
Authors: Szabó Pál Tamás , Kele Zoltán
Published: September 2002

The use of dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) as solvents in electrospray ionization (ESI) is suggested for the analysis of hydrophobic compounds. Its use was shown to overcome solubility problems and resulted in good quality electrospray spectra of protected hydrophobic peptides, sugars and other hydrophobic compounds. Intense protonated and/or sodiated molecules were formed in positive ion mode while negative ion mode resulted in [M + 95]− ions from DMSO and [M + Cl]− ions from DMF in cases where no significant molecular ion related peaks could be observed applying commonly used protic solvents such as methanol or acetonitrile. Deuterium labeling (d6‐DMSO), high resolution experiments and tandem mass spectrometric measurements showed that the methanesulfonic acid (MSA), present in DMSO as a common impurity, is responsible for the formation of protonated molecules in positive ion mode and for methane sulfonate anion adducts [M + 95]− in negative ion mode. Copyright © 2001 John Wiley & Sons, Ltd.
Determination of hydroxyalkyl derivatives of cobalamin (vitamin B12) using reversed phase high performance liquid chromatography with electrospray tandem mass spectrometry and ultraviolet diode array detection
Authors: Alsberg Tomas , Minten Johanna , Haglund Johanna , Törnqvist Margareta
Published: September 2002

Electrospray ionization tandem mass spectrometry (ESI‐MS/MS) and ultraviolet diode array detection (UV‐DAD), coupled on‐line to reversed phase high performance liquid chromatography (HPLC), was used for the characterization of hydroxyalkyl derivatives of cob(I)alamin. The reduced form of vitamin B12, cob(I)alamin, denoted a supernucleophile due to its high nucleophilic strength, has shown promise as an analytical tool in studies of electrophilically reactive compounds in vitro and in vivo. A method for analysis of DNA‐phosphate adducts was developed earlier utilizing the supernucleophilicity of cob(I)alamin to transfer alkyl groups from the phosphotriester configuration in DNA, with the formation of a Co‐substituted alkyl‐cobalamin (alkyl‐Cbl) complex. For the purpose of identification and quantification of alkyl‐Cbls at high sensitivity, an MS/MS method has been developed with application to a number of 2‐hydroxyalkyl‐cobalamins (OHalkyl‐Cbls). The precursor oxiranes were reacted with cob(I)alamin, followed by clean‐up and mass spectrometric analysis of the resulting OHalkyl‐Cbls. It was found that ionization was highly dependent on solvent composition. By using acetonitrile/water/trifluoroacetic acid (TFA) (eluent I), the base peak was the doubly protonated molecule [M + 2H]2+, whereas acetonitrile/water/1‐methylpiperidine (eluent II) yielded the singly protonated molecule [M + H]+ as the base peak. Excellent separation was obtained with eluent II, with good separation between stereoisomers, thus enabling the characterization of these by means of UV spectra. Limits of quantitation for 2‐hydroxypropyl‐cobalamin (OHPr‐Cbl) were 0.2 and 2 pg/µL (or 0.1 and 1 fmol/µL) using selected ion recording (SIR) with eluent I and II, respectively. The obtained detection level should be sufficient for analysis of alkyl‐Cbls from a wide range of toxicological applications. Copyright © 2001 John Wiley & Sons, Ltd.
Serum protein profiles of patients with pancreatic cancer and chronic pancreatitis: searching for a diagnostic protein pattern
Authors: Valerio A. , Basso D. , Mazza S. , Baldo G. , Tiengo A. , Pedrazzoli S. , Seraglia R. , Plebani M.
Published: September 2002

In this study, 13 sera from patients with pancreatic cancer, 9 from chronic pancreatitis and 10 from healthy subjects were analyzed by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. The MALDI mass spectra revealed the presence of several low molecular weight peptides, among which some were detected only in the sera from both pathological conditions. On the other hand many peptides were observed only in control sera, and were absent in the sera from the two diseases. Therefore, MALDI analysis of the low molecular weight fraction (<10 000 Da) of sera from patients with pancreatic diseases enabled us to identify the presence of some disease‐related signals and also some signals characteristic of normal subjects. Copyright © 2001 John Wiley & Sons, Ltd.
A new method to determine the 17O isotopic abundance in CO2 using oxygen isotope exchange with a solid oxide
Authors: Assonov S.S. , Brenninkmeijer C. A. M.
Published: September 2002

This paper discusses a simple method to determine 17O isotope excess or deficiency (‘mass‐independent isotopic composition’) in CO2 gas. When applying conventional mass spectrometry of CO2 (m/z 44, 45 and 46) to determine the 17O/16O ratio, the 13C/12C ratio has to be established separately. This can be achieved by analysing an aliquot of sample CO2 before and after subjecting it to oxygen isotope exchange with a pool of oxygen with ‘normal’ 17O/16O ratio, i.e. with Δ17O≡δ17O−0.516 × δ18O = 0. Cerium oxide has been shown to be practically well suited for the exchange of CO2 oxygen; the reagent is safe and does not produce any contamination. The CO2‐CeO2 exchange reaction has 99.8 ± 0.7% recovery yield. At 650 °C this reaction reaches equilibrium in 30 min and, as tested, results in complete oxygen replacement. Δ17O determinations depend on accuracy of CO2 delta measurements: the repeatability of ±0.015‰ (1σ) in δ45R and δ46R deter‐mination relative to the working reference results in an error of Δ17O as small as ±0.33‰. Such a precision is sufficient for Δ17O determination in stratospheric CO2. The calculated Δ17O value systematically depends on absolute 17R and 13R ratios in isotopic reference materials, which are presently not yet known with certainty (the 17R value is most important), and may be inadequate for 17O‐correction with a = 0.516. Within the present uncertainty, Δ17O determined in 17O‐enriched CO2 agrees with the value directly measured in the enriched O2 from which this CO2 was produced. Besides Δ17O determination, investigated CO2‐CeO2 equilibration may have several other implications. Fast, complete isotopic exchange of CO2 by reaction with CeO2 may also be employed to get reproducible 17O‐correction and, hence, to better monitor small δ13C shifts and to isotopically equilibrate mixtures of CO2 gases. Copyright © 2001 John Wiley & Sons, Ltd.
Formation of binary alloy cluster ions from group‐14 elements and cobalt and comparison with solid‐state alloys
Authors: Zhang Xia , Li Guoliang , Xing Xiaopeng , Zhao Xiang , Tang Zichao , Gao Zhen
Published: September 2002

By using laser ablation on mixtures of transition metal cobalt and group‐14 elements, binary alloy cluster anions were produced while no binary alloy cluster cations were detected, and the homocluster cations of group‐14 elements appeared at very low abundance. The differences between clustering abilities of germanium, tin and lead with cobalt are described, and the chemical bonds in the binary alloy cluster anions appear to indicate a transition from covalent to metal bonds. The cluster anion [CoPb10]− appears in very high abundance (magic number), and an endohedral structure is proposed for this cluster. The cluster anion [CoPb12]−, also representing a magic number, probably has an icosahedral structure. Compared with solid‐state Co/Ge binary alloys, the compositions of most binary alloy cluster anions are germanium‐rich, in which the covalent bonds are predominant. Copyright © 2001 John Wiley & Sons, Ltd.
Determination of trace quaternary ammonium surfactants in water by combining solid‐phase extraction with surface‐assisted laser desorption/ionization mass spectrometry
Authors: Chen Yu‐Chie , Sun Mei‐Chuan
Published: September 2002

This study demonstrates the feasibility of combining solid‐phase extraction (SPE) with surface‐assisted laser desorption/ionization (SALDI) mass spectrometry to determine trace quaternary ammonium surfactants in water. The trace surfactants in water were directly concentrated on the surface of activated carbon sorbent in SPE. The activated carbon sorbent was then mixed with the SALDI liquid for SALDI analysis. No SPE elution procedure was necessary. Experimental results indicate that the surfactants with longer chain alkyl groups exhibit higher sensitivities than those with shorter chain alkyl groups in SPE‐SALDI analysis. The detection limit for hexadecyltrimethylammonium bromide is around 10 ppt in SPE‐SALDI analysis by sampling 100 mL of aqueous solution, while that of tetradecyltrimethylammonium bromide is about 100 ppt. The detection limit for decyltrimethylammonium bromide and dodecyltrimethylammonium bromide is in the low‐ppb range. Copyright © 2001 John Wiley & Sons, Ltd.
Electron ionisation induced fragmentation of ethyl 5(1H)‐oxo‐ and 7(1H)‐oxo‐1‐aryl‐2,3‐dihydroimidazo[1,2‐a]‐pyrimidine‐6‐carboxylates: evidence for an unusually regioselective rearrangement of M+• ions
Authors: Ovcharenko Vladimir V. , Pihlaja Kalevi , Matosiuk Dariusz
Published: September 2002

The 70‐eV electron ionisation (EI) mass spectra of the title compounds show clear differences between the 5‐oxo and 7‐oxo isomers due to regioselective fragmentations involving the ester function. Exceptionally abundant metastable peaks due to molecular ions fragmenting to [M −CO2]+· were observed exclusively for the 7‐oxo isomers, suggesting that the sufficiently long‐lived molecular ions undergo a slow rearrangement preceding this fragmentation reaction. The results are contrasted to the available literature data on the ester group fragmentations involving the loss of CO2 and the EI mass spectrometry of pyrimidone β‐oxo esters. A reaction mechanism is proposed for the elimination of CO2 following ethyl group migration to the pyrimidone carbonyl oxygen. Copyright © 2001 John Wiley & Sons, Ltd.
Determination of midazolam in human plasma by solid‐phase microextraction and gas chromatography/mass spectrometry
Authors: Frison Giampietro , Tedeschi Luciano , Maietti Sergio , Ferrara Santo Davide
Published: September 2002

A new method is described for the qualitative and quantitative analysis of midazolam, a short‐acting 1,4‐imidazole benzodiazepine, in human plasma. It involves a plasma deproteinization step, solid‐phase microextraction (SPME) of midazolam using an 85‐µm polyacrylate fiber, and its detection by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode, using pinazepam as internal standard. The assay is linear over a midazolam plasma range of 1.5–300 ng/mL, relative intra‐ and inter‐assay standard deviations at 5 ng/mL are below 7%, and the limit of detection is 1 ng/mL. The method is simple, fast and sufficiently sensitive to be applied in clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring. Copyright © 2001 John Wiley & Sons, Ltd.
Gas chromatography/mass spectrometry demonstration of steryl glycosides in eucalypt wood, Kraft pulp and process liquids
Authors: Gutiérrez Ana , del Río José C.
Published: September 2002

The occurrence of steryl glycosides (SG) and acyl steryl glycosides (ASG) in eucalypt (Eucalyptus globulus) wood has been investigated. These compounds were analyzed as their trimethylsilyl ethers by gas chromatography/mass spectrometry (GC/MS) using a 15 m length high‐temperature capillary column with a thin film, and identified on basis of their mass spectra and relative retention times comparing with those of authentic standards. Significant amounts of SG were identified in eucalypt wood whilst only traces of ASG could be detected. Eucalypt SG and ASG occur in the pyranoside form, which is readily distinguishable from the furanoside configuration by the mass spectra of their trimethylsilyl derivatives. The sterol part of the SG and ASG consisted of sitosterol, being sitosteryl 3β‐D‐glucopyranoside and sitosteryl (6′‐O‐palmitoyl)‐3β‐D‐glucopyranoside, the major SG and ASG found in E. globulus wood. The presence of SG and ASG was also investigated after kraft cooking by analyzing unbleached pulp and process water samples. The GC/MS results also revealed the presence of sitosteryl 3β‐D‐glucopyranoside in these samples. By contrast, no ASG could be detected. Therefore, we have shown that SG survive the kraft cooking and can be found at least partly intact after pulping, being a possible cause for pitch deposits together with free and esterified sterols. Copyright © 2001 John Wiley & Sons, Ltd.
Gas chromatography/combustion/isotope ratio mass spectrometry to control the misuse of androgens in breeding animals: new derivatisation method applied to testosterone metabolites and precursors in urine samples
Authors: Prévost Stéphanie , Nicol Tatiana , Monteau Fabrice , André François , Bizec Bruno Le
Published: September 2002

A new derivatisation reaction applied to the analysis of steroids by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) was studied. The trimethylsilylated steroids were characterised by well‐resolved chromatographic signals, no peak tailing, reproducible 13C/12C measurements (0.32‰, n = 28), good signal‐to‐noise ratio and absolute intensity (5 × 10−9 A, 20 ng), and a slow degradation of copper oxide pellets in the combustion furnace. In addition, two new metabolites and one precursor of testosterone in bovine have been brought into consideration and used for GC/C/IRMS measurements, namely, 3β‐hydroxy‐5α‐androstan‐17‐one (epiandrosterone), 3β,17α‐dihydroxy‐5α‐androstane, and 3β,17α‐dihydroxy‐5‐androstene. The new findings have been applied to an elimination study in bovine of testosterone metabolites after an intramuscular injection of testosterone enanthate. Significant differences (up to 4‰) between testosterone metabolites and precursor were detectable at least three weeks after administration. Copyright © 2001 John Wiley & Sons, Ltd.