Molecular Mass Measurements Research Articles

Application of complementary mass spectrometric techniques to the identification of ketoprofen phototransformation products

Ketoprofen (KP) is a nonsteroidal anti-inflammatory drug, which during UV irradiation rapidly transforms into benzophenone derivatives. Such transformation products may occur after topical application of KP, which is then exposed to sunlight resulting in a photo-allergic reaction. These reactions are mediated by the benzophenone moiety independently of the amount of allergen. The same reactions will also occur during wastewater or drinking water treatment albeit their effect in the aqueous environment is yet to be ascertained. In addition, only a few such transformation products have been recognised. To enable the detection and structural elucidation of the widest range of KP transformation products, this study applies complementary chromatographic and mass spectrometric techniques including gas chromatography coupled to single quadrupole or ion trap mass spectrometry and liquid chromatography hyphenated with quadrupole-time-of-flight mass spectrometry. Based on structural information gained in tandem and multiple MS experiments, and on highly accurate molecular mass measurements, chemical structures of 22 transformation products are proposed and used to construct an overall breakdown pathway. Among the identified transformation products all but two compounds retained the benzophenone moiety--a result, which raises important issues concerning the possible toxic synergistic effects of KP and its transformation products. These findings trigger further research into water treatment technologies that would limit their entrance into environmental or drinking waters.

CYP2D6 genotyping by liquid chromatography-electrospray ionization mass spectrometry

Genetic polymorphisms can significantly affect the enzyme activity of the drug metabolizing enzyme Cytochrome P450 2D6 (CYP2D6; OMIM 124030). Accordingly, CYP2D6 genotyping is considered as a valid approach to predict the individual CYP2D6 metabolizing status. We introduce ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) as method for the characterization of single base variants, small deletions, and insertions in the CYP2D6 gene. A two-step polymerase chain reaction (PCR) was developed for the simultaneous amplification of nine polymorphic regions within the CYP2D6 gene. Cleanup, separation, and denaturation of PCR amplicons were achieved by high-performance liquid chromatography. High-performance molecular mass measurements provided nucleotide composition profiles that principally enable the resolution of 37 reported CYP2D6 alleles. The developed assay was applied to the genotyping of 93 unrelated Austrian individuals. For validation, a selected number of samples and polymorphic sites were retyped by alternative genotyping technologies. The PCR-ICEMS assay turned out to be an accurate, robust, and cost-effective CYP2D6 genotyping strategy.

The Subunit Composition of Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) From Pichia pastoris

Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-α-formyl methionine residues. At least eight subunits are N-α-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components.

Extensive De Novo Sequencing of New Parvalbumin Isoforms Using a Novel Combination of Bottom-Up Proteomics, Accurate Molecular Mass Measurement by FTICR−MS, and Selected MS/MS Ion Monitoring

Parvalbumins (PRVB) (11.20-11.55 kDa) are considered the major fish allergens. In this work, we propose a novel strategy for extensive characterization of this group of proteins based on the integration of a classical Bottom-Up proteomics approach with accurate Mr determination by FTICR-MS of intact proteins and selected MS/MS ion monitoring (SMIM) of peptide mass gaps. For each PRVB, mass spectra obtained by LC-ESI-IT-MS/MS from two digests (trypsin, Glu-C) were de novo sequenced manually with help of two programs (PEAKS, DeNovoX). The deduced peptide sequences were arranged and the theoretical Mr for the resulting sequences was calculated. Experimental Mr for each PRVB was measured with high mass accuracy by FTICR-MS (0.05-4.47 ppm). The masses of several missing peptide gaps were estimated by comparing the theoretical and experimental Mr, and the MS/MS spectra corresponding to these ions were obtained by LC-ESI-IT-MS/MS in the SMIM scanning mode. Finally, all peptide sequences were combined to generate the final protein sequences. This approach allowed the complete de novo MS-sequencing of 25 new PRVB isoforms. These new sequences belong to 11 different species from the Merlucciidae family, organisms for which genomes remain unsequenced. This study constitutes the report accounting for the higher number of new proteins completely sequenced making use of MS-based techniques only.

Tandem mass spectrometric de novo sequencing of oligonucleotides using simulated annealing for stochastic optimization

A global strategy for tandem mass spectrometric de novo sequencing of oligonucleotides is presented which is based on finding among all possible sequences the sequence, whose simulated tandem mass spectrum shows the highest degree of similarity to the measured spectrum. Global de novo sequencing can become a time-consuming task due the exponential increase of the number of possible sequences with increasing oligonucleotide length. For the reduction of sequencing effort and time, simulated annealing was introduced as stochastic optimization technique that is capable to find the optimal sequence by testing only a subset of all possible configurations. We have evaluated the performance of the de novo sequencing approach by sequencing 13 synthetic oligonucleotides ranging in length from 5 to 24 nucleotides. Mass spectrometric and tandem mass spectrometric experiments were performed on a quadrupole-quadrupole-time-of-flight instrument. Accurate molecular mass measurements were used to determine the nucleotide compositions of the oligonucleotides. For the majority of tested oligonucleotides two different charge states were selected as precursor ions. Each precursor ion was fragmented applying several different collision voltages. Overall 107 fragment ion mass spectra were acquired. The nucleotide compositions together with the tandem mass spectral information represented the input for the de novo sequencing algorithm. Sequences of oligonucleotides as large as 22-mers were correctly determined in more than 90% of cases. The optimal sequence was retrieved within 1–2min. Fragment ion mass spectra of larger oligonucleotides were inappropriate for de novo sequencing. The occurrence of extensive internal fragmentation causing low sequence coverage paired with a high probability of assigning fragment ions to wrong sequences gave rise to incorrect results.

Determination of the Number Average Molecular Mass of Asphaltenes (Mn) Using Their Soluble A2 Fraction and the Vapor Pressure Osmometry (VPO) Technique

Fractions A1 and A2 have been isolated from asphaltenes using the para-nitrophenol (PNP) method,(20, 21) with fraction A2 being characterized as one with a tendency for aggregation smaller than asphaltenes and the opposite being observed for fraction A1. Hence, measuring molecular mass (MM) properties of fraction A2 would suggest MM properties of asphaltenes where aggregation is either smaller or insignificant. In this work, using several samples of asphaltenes and different solvents and temperatures, we investigated the aggregation tendency of fraction A2 using the vapor pressure osmometry (VPO) technique. When measurement of the number average molecular mass (Mn) was performed in toluene and o-dichlorobenzene (ODB), Mn ≈ 1000 g mol−1 was found. Evidence for fraction A2 aggregation was detected in chloroform and nitrobenzene. We found that ODB was the best solvent for VPO, where a constant value close to 1000 g mol−1 was measured for fraction A2 when the temperature was changed from 80 to 120 °C. In all ...

Quality control of protein standards for molecular mass determinations by small-angle X-ray scattering

Small-angle X-ray scattering (SAXS) is a powerful technique with which to evaluate the size and shape of biological macromolecules in solution. Forward scattering intensity normalized relative to the particle concentration,I(0)/c, is useful as a good measure of molecular mass. A general method for deducing the molecular mass from SAXS data is to determine the ratio ofI(0)/cof a target protein to that of a standard protein with known molecular mass. The accuracy of this interprotein calibration is affected considerably by the monodispersity of the prepared standard, as well as by the precision in estimating its concentration. In the present study, chromatographic fractionation followed by hydrodynamic characterization is proposed as an effective procedure by which to prepare a series of monodispersed protein standards. The estimation of molecular mass within an average deviation of 8% is demonstrated using monodispersed bovine serum albumin as a standard. The present results demonstrate the importance of protein standard quality control in order to take full advantage of interprotein calibration.

Synthesis, characterization, and application of nanoporous materials based on silicon- or halogen-containing spiroketal and spirothioketal polymers

Organic microporous materials based on silicon-containing spiroketal and spirothioketal polymers were synthesized via a 1,3-dioxol-forming polymerization reaction between 1,1a,4,4a,5,5a,8, 8a-octahydro-2,3,6,7-tetra(trimethylsilyl)-9,10-anthraquinone and different types of polyol or polythiol. These silicon-containing polymers were subjected to bromination to yield bromine-containing polymers. The structures of the prepared polymers were confirmed by NMR spectroscopy and molecular mass measurements. Nitrogen adsorption/desorption isotherms of the prepared polymers showed that a large amount of nitrogen was adsorbed at low relative pressure, indicating microporosity. These polymers have BET surface areas in the range of 505-830 m^2 g^{-1}. These polymers were determined to be useful for pervaporation separation of phenol/water mixtures.

Validation of molecular mass measurements by means of diffusion-ordered NMR spectroscopy: Application to oligosaccharides

Abstract The validation of molecular mass measurements by using DOSY spectrocopy is presented. A mixture of oligosaccharides has been studied and an extended model has been used. A method has been proposed and applied to correct the imperfections due either to the lock system or the room temperature regulator.

Treatise on the measurement of molecular masses with ion mobility spectrometry.

The ability to separate isotopes by high-resolution ion mobility spectrometry techniques is considered as a direct means for determining mass at ambient pressures. Calculations of peak shapes from the transport equation show that it should be possible to separate isotopes for low-mass ions (<200) by utilizing heavy collision gases and high-resolution ion mobility analyzers. The mass accuracy associated with this isotopic separation approach based on ion mobility separation is considered. Finally, we predict several isotopes that should be separable.

Proton‐induced crosslinking of CR 6‐2 polycarbonate

AbstractCR 6‐2 polycarbonate samples were irradiated using different fluences (1011–1014 ions/cm2) of 1 MeV protons. The structural modifications in the proton‐irradiated CR 6‐2 samples have been studied as a function of fluence using different characterization techniques such as X‐ray diffraction, intrinsic viscosity of the liquid samples, as a measure of the mean molecular mass, Fourier transform infrared spectroscopy, thermogravimetric analysis, differential thermal analysis, refractive index, color difference, and mechanical properties. The results indicate that the irradiation of CR 6‐2 detector at the fluence range 1 × 1012–5 × 1014 ions/cm2 causes intermolecular crosslinking and allows the formation of covalent bonds between different chains, leading to a more compact structure of CR 6‐2 polymer, which resulted in an improvement in its thermal, optical, and mechanical properties. Also, this crosslinking reduces the ordering structure and increases the amorphous regions that enhance the polymer resilience. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Effect of neutron dose on the structural properties of Makrofol polycarbonate

The effect of neutron dose on the structural properties of a Makrofol polycarbonate detector has been studied. Samples of Makrofol were classified into three main groups. The first, second and third groups were irradiated with neutrons in the dose ranges of 0.01–0.08, 0.1–0.8 and 1.0–8.0 Sv, respectively. The structural modifications in the neutron irradiated samples were studied using X-ray diffraction and Fourier-transform infrared spectroscopy. In addition, the effect of neutron dose on the intrinsic viscosity of the liquid samples, as a measure of the mean molecular mass of the Makrofol polymer, was studied. An increase in the –OH groups was observed at dose ranges of 0.01–0.04 and 3.21–8.0 Sv due to the degradation of carbonate group and the –H abstraction from the polymer backbone to form hydroxyl groups. This indicates that the degradation is dominant at these dose ranges that enhance the degree of ordering in the irradiated samples. On the other hand, irradiation in the dose range 0.04–3.21 Sv was characterized by a dominant crosslinking mechanism that led to an increase of the amorphous phase and average molecular mass.

Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for G N -G C Glycoprotein Heterodimers

Rift Valley fever virus (RVFV) is a member of the genus Phlebovirus within the family Bunyaviridae. It is a mosquito-borne zoonotic agent that can cause hemorrhagic fever in humans. The enveloped RVFV virions are known to be covered by capsomers of the glycoproteins G(N) and G(C), organized on a T=12 icosahedral lattice. However, the structural units forming the RVFV capsomers have not been determined. Conflicting biochemical results for another phlebovirus (Uukuniemi virus) have indicated the existence of either G(N) and G(C) homodimers or G(N)-G(C) heterodimers in virions. Here, we have studied the structure of RVFV using electron cryo-microscopy combined with three-dimensional reconstruction and single-particle averaging. The reconstruction at 2.2-nm resolution revealed the organization of the glycoprotein shell, the lipid bilayer, and a layer of ribonucleoprotein (RNP). Five- and six-coordinated capsomers are formed by the same basic structural unit. Molecular-mass measurements suggest a G(N)-G(C) heterodimer as the most likely candidate for this structural unit. Both leaflets of the lipid bilayer were discernible, and the glycoprotein transmembrane densities were seen to modulate the curvature of the lipid bilayer. RNP densities were situated directly underneath the transmembrane densities, suggesting an interaction between the glycoprotein cytoplasmic tails and the RNPs. The success of the single-particle averaging approach taken in this study suggests that it is applicable in the study of other phleboviruses, as well, enabling higher-resolution description of these medically important pathogens.

NMR Measure of Translational Diffusion and Fractal Dimension. Application to Molecular Mass Measurement

Experimental NMR diffusion measure on polymers and on globular proteins are presented. These results, complemented with results found in the literature, enable a general description of effective fractal dimension for objects such as small organic molecules, sugars, polymers, DNA, and proteins. Results are compared to computational simulations as well as to theoretical values. A global picture of the diffusion phenomenon emerges from this description. A power law relating molecular mass with diffusion coefficients is described and found to be valid over 4 orders of magnitude. From this law, the fractal dimension of the molecular family can be measured, with experimental values ranging from 1.41 to 2.56 in full agreement with theoretical approaches. Finally, a method for evaluating the molecular mass of unknown solutes is described and implemented as a Web page.

Unraveling the metabolic transformation of tetrazepam to diazepam with mass spectrometric methods

The metabolic transformation pathways of the 1,4-benzodiazepine tetrazepam (C(16)H(17)ClN(2)O, average mass: 288.772) were studied with capillary LC-QqTOF-MS and -MS/MS by analyzing human plasma and urine samples collected from healthy volunteers. Each volunteer took 50 mg of tetrazepam, given in the form of one tablet of Myolastan (Sanofi-Synthelabo, Vienna, Austria). Accurate molecular mass measurements in full-scan mode (scan range: 50-700) were used to survey the collected samples for putative metabolic transformation products. Full-scan fragment ion mass spectra were collected in subsequent LC/MS/MS experiments. Each spectrum was matched to a spectral library containing 3759 MS/MS-spectra of 402 compounds, including eighteen different benzodiazepines, to prove the structural relatedness of a tentative metabolite to tetrazepam. This "similarity search" approach provided a rapid and powerful tool to exclude non-drug-related species, even without any knowledge of the fragmentation chemistry. Interpretation of tandem mass spectrometric data was only required in order to elucidate the site of transformation. Using this strategy, 11 major classes of tetrazepam metabolites were identified. Possible metabolic routes from tetrazepam to diazepam (C(16)H(13)ClN(2)O, average mass: 284.740) via repeated hydroxylation and dehydration of the cylohexenyl moiety were discovered. No evidence for extensive hydroxylation of tetrazepam at position 3 of the diazepine ring was found. In contrast to what is commonly believed, this distinct transformation reaction may be of only minor importance. Furthermore, the occurrence of demethylation, hydration, and glucuronidation reactions was proven.

Analysis of a G protein-coupled receptor for neurotensin by liquid chromatography–electrospray ionization–mass spectrometry

The type 1 neurotensin receptor (NTS1) belongs to the G protein-coupled receptor (GPCR) family. GPCRs are involved in important physiological processes, but for many GPCRs ligand binding sites and other structural features have yet to be elucidated. Comprehensive analyses by mass spectrometry (MS) could address such issues, but they are complicated by the hydrophobic nature of the receptors. Recombinant NTS1 must be purified in the presence of detergents to maintain solubility and functionality of the receptor, to allow testing of ligand, or to allow G protein interaction. However, detergents are detrimental to MS analyses. Hence, steps need to be taken to substitute the detergents with MS-compatible polar/organic solvents. Here we report the characterization of NTS1 by electrospray ionization (ESI)–MS with emphasis on methods to transfer intact NTS1 or its proteolytic peptides into compatible solvents by protein precipitation and liquid chromatography (LC) prior to ESI–MS analyses. Molecular mass measurement of intact recombinant NTS1 was performed using a mixture of chloroform/methanol/aqueous trifluoroacetic acid as the mobile phase for size exclusion chromatography–ESI–MS analysis. In a separate experiment, NTS1 was digested with a combination of cyanogen bromide and trypsin and/or chymotrypsin. Subsequent reversed phase LC–ESI–tandem MS analysis resulted in greater than 80% sequence coverage of the NTS1 protein, including all seven transmembrane domains. This work represents the first comprehensive analysis of recombinant NTS1 using MS.

Self-organizing cyclolinear methylcyclohexasiloxane polymers carrying reactive vinyl groups

Atactic cyclolinear organosilicon polymers containing vinyl substituents in RSiO1.5, R2SiO or both moieties have been synthesized through the heterofunctional polycondensation of trans,cis-2,8-dihydroxymethyl(vinyl)cyclohexasiloxanes with 2,8-dichloromethyl(vinyl)cyclohexasiloxanes. The structure of the polymers has been studied by 1H and 29Si NMR and IR spectroscopy, molecular mass measurements, and elemental analysis. The phase behavior of these copolymers in the bulk has been examined by DSC, X-ray diffraction, and polarization optical microscopy. It has been shown that the copolymer can exist in the mesomorphic state in the temperature range from −100 to +200°C. The X-ray data indicate changes in the interlayer spacing and the type of packing of cyclolinear poly(methylvinylsiloxanes) with an increase in the content of vinyl substituents in the repeating units of the polymer. The ability of cyclolinear poly(methylvinylsiloxanes) to spread over the water/air interface and to form mono-and multilayers has been investigated. As the content of vinyl substituents in the polymer unit is increased to two or four, the ability of polymers to form multilayers is preserved. The incorporation of vinyl substituents into RSiO1.5 or R2SiO moieties of polymer units is accompanied by the formation of monolayers.

Identification and human pharmacokinetics of dihydroergotoxine metabolites in man: preliminary results

Dihydroergotoxine is a mixture of semi-synthetic ergot alkaloids mainly used for age-related cognitive impairment. In this study, dihydroergotoxine (30 microM) was added to incubates of rat and bovine liver microsomes, and the resulting major metabolites were identified as hydroxy-dihydroergocornine, hydroxy-dihydroergocryptine and hydroxy-dihydroergocristine on the basis of molecular mass measurements, determined with a time-of-flight mass spectrometer. The relevance of these to humans was then investigated by simultaneously monitoring dihydroergotoxine and its hydroxy-metabolites in human plasma by LC-MS/MS after oral dosing of dihydroergotoxine mesylate (27 mg) to a healthy volunteer (male, age 45, height 1.93 m, weight 103 kg). In this preliminary approach, the peaks (C(max)) of dihydroergocornine, dihydroergocryptine and dihydroergocristine were about 0.04 microg/l. The peaks (C(max)) of their hydroxy-metabolites were 0.98, 0.53 and 0.30 microg/l, respectively. In conclusion, in this preliminary approach it was found that hydroxy-dihydroergocornine, hydroxy-dihydroergocryptine and hydroxy-dihydroergocristine were one order of magnitude higher in concentration than their parents in human plasma.

The c-Myc Target Gene Rcl (C6orf108) Encodes a Novel Enzyme, Deoxynucleoside 5′-monophosphate N-Glycosidase

RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5'-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5'-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.

On the Proper Use of Mass Accuracy in Proteomics

Mass measurement is the main outcome of mass spectrometry-based proteomics yet the potential of recent advances in accurate mass measurements remains largely unexploited. There is not even a clear definition of mass accuracy in the proteomics literature, and we identify at least three uses of this term: anecdotal mass accuracy, statistical mass accuracy, and the maximum mass deviation (MMD) allowed in a database search. We suggest using the second of these terms as the generic one. To make the best use of the mass precision offered by modern instruments we propose a series of simple steps involving recalibration of the data on "internal standards" contained in every proteomics data set. Each data set should be accompanied by a plot of mass errors from which the appropriate MMD can be chosen. More advanced uses of high mass accuracy include an MMD that depends on the signal abundance of each peptide. Adapting search engines to high mass accuracy in the MS/MS data is also a high priority. Proper use of high mass accuracy data can make MS-based proteomics one of the most "digital" and accurate post-genomics disciplines.