Biochemical Characterization of a Uranyl Ion‐Specific DNAzyme

Uranyl ion‐specific DNAzyme : A DNAzyme (lower strand) cleaves the substrate (upper strand) in the presence of the uranyl ion. The enzyme folds into a bulged three‐way‐junction structure with catalytically important nucleotides residing in the bulge. A highly conserved G?A mismatch is also crucial for the enzyme's activity.

     

Comparative 13C‐NMR and matrix‐assisted laser desorption/ionization time‐of‐flight analyses of species variation and structure maintenance during melamine–urea–formaldehyde resin preparation

The preparation of an industrially used sequential formulation of a melamine–urea–formaldehyde resin was followed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and ¹³C‐NMR analysis. The analysis allowed us to identify and follow the appearance, increase, decrease, and disappearance of a multitude of chemical species during the preparation of both the initial urea–formaldehyde (UF) phase of the reaction and the subsequent reaction of melamine with the UF resin that formed. The analysis indicated that (1) the increase and decrease in the species that formed proceeded through a cycle of the formation and degradation of species occurring continuously through what appeared to be a series of complex equilibria, (2) even at the end of the reaction a predominant proportion of methylene ether bridges was still present, (3) some small proportion of methylene bridges already had formed in the UF reaction phase of the resin even under rather alkaline conditions, and (4) the addition of melamine to the UF prepolymer induced some noticeable rearrangement of methylene ether bridges to methylene bridges. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

Fully Enzymatic N→C‐Directed Peptide Synthesis Using C‐Terminal Peptide α‐Carboxamide to Ester Interconversion

Chemoenzymatic peptide synthesis is potentially the most cost‐efficient technology for the synthesis of short and medium‐sized peptides with some important advantages. For instance, stoichiometric amounts of expensive coupling reagents are not required and racemisation does not occur rendering purification easier compared to chemical peptide synthesis. In this paper, a novel interconversion reaction of peptide C‐terminal α‐carboxamides into primary alkyl esters with alcalase was used to develop a fully enzymatic peptide synthesis strategy. For each elongation step a cost‐efficient amino acid carboxamide building block was used followed by the interconversion of the elongated peptide carboxamide to the corresponding primary alkyl ester. These peptide esters are the starting materials for the next enzymatic peptide elongation step.     

Molar mass distributions of polymers from size exclusion chromatography and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: Methods for comparison

Equations are presented for calculating molar mass averages and molar mass distributions from matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) data and from size exclusion chromatography (SEC) data. The utility of polydispersity is examined as an indicator of the expectation of MALDI‐TOF MS mass discrimination effects. Cumulative distributions are found to be rich in information for comparing the two techniques and are easily obtained from both SEC and MALDI‐TOF MS data. Analyses of a series of narrow molar mass distribution poly(methyl methacrylate) (PMMA) standards and one polydisperse sample have been performed with both methods. MALDI‐TOF MS did not detect dimer and trimer in the PMMA samples, and it often indicated lower amounts of high‐molar‐mass polymers than did SEC. The results showed that the distribution breadth, as evidenced by the standard deviation of the distribution (calculated from the polydispersity and number‐average molar mass), correlated well with the molar mass range observed in the MALDI‐TOF MS spectra, whereas the polydispersity alone did not. Ratioing the extremes in the molar mass concentrations measured with the SEC differential refractometer, which were necessary to adequately define molar mass distributions, showed that detector dynamic range values as high as approximately 370,000 were required for the polydisperse samples. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 627–639, 2005     

Time‐of‐flight secondary ion mass spectrometry investigation of epoxy resin curing behavior in real time

Time‐of‐flight secondary ion mass spectrometry and principal components analysis were used in real time to monitor the progress of curing reactions on the surface of a diglycidyl ether of bisphenol A (DGEBA) and diglycidyl ether of bisphenol F (DGEBF) epoxy resin blend reacted with the diamine hardener isophorone diamine at different time intervals. Molecular ions in the mass spectra that characterized the curing reactions steps, including blocking, coupling, branching, and crosslinking, were identified. The aliphatic hydrocarbon ions were correlated to the curing reaction rate, and this indicated that coupling and branching occurred much faster than the blocking and crosslinking curing reactions steps. The total conversion of the coupling and branching reaction steps were followed on the basis of changes with time in the relative ion intensity of molecular ions assigned to the DGEBA/DGEBF, aliphatic hydrocarbon, epoxide, and aromatic ring structures. Indicative measures of crosslinking density were monitored through the observation of changes in the ratio of the relative intensities of the aliphatic hydrocarbon and hydroxyl molecular ions over time. The curing reaction conversion was established by the observation of the changes in the relative ion intensity of the molecular ions that were related to the DGEBA/DGEBF molecules. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

High‐performance liquid chromatography‐mass spectrometry of epoxy resins

The application of liquid chromatography coupled to mass spectrometry (LC‐MS) for the analysis of epoxy resins is shown in two examples. Electro spray (ESI) and atmospheric pressure chemical ionization (APCI) are compared with respect to the ionization of diglycidylether of bisphenol A‐based (DGEBA) epoxy resins. By‐products in a typical modified solid DGEBA‐based epoxy resin and in a new weatherable crosslinker for powder coating applications are characterized and discussed. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 913–925, 1999     

The Biosynthesis of the Aroma Volatile 2‐Methyltetrahydrothiophen‐3‐one in the Bacterium Chitinophaga Fx7914

2‐Methyltetrahydrothiophen‐3‐one ( 3 ) is a volatile compound that plays an important role especially in food and flavour chemistry because it contributes to the aroma of several foodstuffs including wine. Although 3 can be formed by chemical reactions during food preparation, it is also produced by microorganisms. Recent studies with yeasts showed that methionine ( 1 ) is a potential precursor of 3 , but the mechanism of the transformation is unknown. The biosynthetic pathway leading to 3 in the bacterium Chitinophaga Fx7914 was probed. Extensive feeding experiments with differently labelled precursors by using liquid cultures of Chitinophaga Fx7914 were performed. The volatiles released by the bacterium were collected by using a closed loop stripping apparatus (CLSA) and analysed by GC–MS. The observed incorporation pattern of the precursors into 3 led to the elucidation of the biosynthetic pathway. One part of the compound 2 originates from homocysteine ( 15 ), which is transformed into 3‐mercaptopropanal ( 17 ). The second biosynthetic building block is pyruvate ( 14 ). An acyloin‐forming reaction furnishes the key intermediate 21 , which cyclises intramolecularly to a diol. Dehydration followed by tautomerisation lead to the cyclic ketone 3 , which is produced by the bacterium in racemic form.     

A High‐Throughput Mass‐Spectrometry‐Based Assay for Identifying the Biochemical Functions of Putative Glycosidases

The most commonly employed glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position in potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a qualitative mass spectrometry–based glycosidase assay amenable to high‐throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high‐throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved through the observation of an appropriate decrease in mass between a potential sugar substrate and its corresponding product by electrospray ionization mass spectrometry (ESI‐MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.     

Solid‐phase microextraction (SPME) in polymer characterization—Long‐term properties and quality control of polymeric materials

Solid‐phase microextraction (SPME) in combination with GG‐MS was applied to quality control polyamide 6.6 collected for recycling and to study the long‐term properties and degradation of nitrile rubber, polyethylene, and polyamide 6.6. The migration of plasticizer and other additives reduces the service‐life and changes the properties of the material. It is also a possible health hazard, for example, legislation against the use of brominated flame retardants in plastic materials, is under discussion, and fast and reliable methods are required to detect such compounds in plastic materials collected for recycling. SPME rapidly and effectively extracted several brominated compounds from in‐plant collected polyamide 6.6. Migration of tris(2‐butoxyethyl)phosphate plasticizer and its degradation products from nitrile rubber during long‐term thermal ageing at 60 and 80°C was shown by SPME‐GC‐MS, while the plasticizer was not volatile enough to be detected by traditional HS‐GC‐MS. In accordance the number of degradation products extracted from thermo‐oxidized PE by HS‐SPME was three times larger than the number detected after HS‐GC‐MS analysis. SPME‐GC‐MS could also detect early signs of degradation in thermo‐oxidized virgin and in‐plant recycled polyamide 6.6 before any signs of degradation were observed by, for example, tensile testing or FTIR. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 867–873, 2003     

Position‐Specific Mass Shift Analysis: A Systematic Method for Investigating the EI‐MS Fragmentation Mechanism of epi‐Isozizaene

The EI‐MS fragmentation mechanism of the bacterial sesquiterpene epi‐isozizaene was investigated through enzymatic conversion of all 15 synthetic (¹³C₁)FPP isotopomers with the epi‐isozizaene synthase from Streptomyces albus and GC‐MS and GC‐QTOF analysis including MS‐MS. A systematic method, which we wish to call position‐specific mass shift analysis, for the identification of the full set of fragmentation reactions was developed.     

Multi‐phosphorylation of the Intrinsically Disordered Unique Domain of c‐Src Studied by In‐Cell and Real‐Time NMR Spectroscopy

Intrinsically disordered regions (IDRs) are preferred sites for post‐translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase–phosphatase networks. Here we used real‐time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the “unique domain” of c‐Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin‐dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP‐dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK‐dependent sites, thus suggesting indirect effects of kinase–phosphatase networks. This study provides a proof‐of‐concept of the use of real‐time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains.     

One‐Step C‐Terminal Deprotection and Activation of Peptides with Peptide Amidase from Stenotrophomonas maltophilia in Neat Organic Solvent

Chemoenzymatic peptide synthesis is a rapidly developing technology for cost effective peptide production on a large scale. As an alternative to the traditional C→N strategy, which employs expensive N‐protected building blocks in each step, we have investigated an N→C extension route that is based on activation of a peptide C‐terminal amide protecting group to the corresponding methyl ester. We found that this conversion is efficiently catalysed by Stenotrophomonas maltophilia peptide amidase in neat organic media. The system excludes the possibility of internal peptide cleavage as the enzyme lacks intrinsic protease activity. The produced peptide methyl ester was used for peptide chain extension in a kinetically controlled reaction by a thermostable protease.

     

Deciphering the structure of itaconate‐based unsaturated polyester resins by high resolution mass spectrometry

Unsaturated polyester (UP) resins are widely used to manufacture composite materials and fulfil a wide panel of specification for industrial or domestic applications at low cost. These resins consist of a highly viscous polyester oligomer and a reactive diluent, which allow their processability and crosslinking. The oligomers are synthesized from diols and saturated or unsaturated diacids. Maleic anhydride is classically used as an unsaturated acid because of its reactivity for esterification and its competitive cost. However, maleic anhydride is petroleum based and classified as skin and respiratory sensitizing by the European Chemicals Agency. Itaconic acid which is recognized as one of the top 12 biobased molecules by the US Department of Energy is a biobased alternative with a reactive unsaturation. In this work, a UP based on propylene glycol, itaconic acid and dimethyl terephthalate was synthesized by polycondensation and characterized by high resolution mass spectroscopy, ¹H, ¹³C 1D and 2D NMR as well as ion mobility spectrometry mass spectrometry. The main structures were linear but cyclic species and a few branched chains due to the intermolecular Ordelt reaction were also detected by NMR and high resolution mass spectroscopy. Molecular modelling has indeed demonstrated that itaconate‐based UPs are more prone to cyclization than fumarate‐based UPs. Moreover, NMR analysis showed a significant consumption of itaconate unsaturation by the Ordelt reaction. Ion mobility spectrometry mass spectrometry allowed an additional structure to be distinguished for the same m/z peaks first attributed to a linear structure only. These results suggest the existence of cyclic species based on an intramolecular Ordelt reaction. © 2020 Society of Chemical Industry     

Dual Maleimide Tagging for Relative and Absolute Quantitation of Cysteine‐Containing Peptides by MALDI‐TOF MS

A dual maleimide (DuMal) tagging method has been developed for both relative and absolute quantitation of cysteine‐containing peptides (CCPs) in combination with MALDI‐TOF mass spectrometry. A pair of maleimides with minimal differences in their chemical structures, N‐methylmaleimide and Nethylmaleimide, have been chosen to allow for the rapid (≈minutes) tagging of CCPs in the Michael addition reaction with high efficiency. It has been validated that the DuMal tagging technique is sensitive and reliable in the quantitative analysis of CCPs. Absolute quantitation of CCPs can be achieved with a detection limit as low as 7.3 nm . Relative quantitation of CCPs can be performed in various sample mixtures with consistent results (coefficient of variation <5 %). The DuMal tagging technique provides a sensitive and accurate approach for the quantitation of biomolecules containing thiol reactive sites; thus it is promising for protein detection, disease diagnosis, and biomarker discovery associated with post‐translational modifications of cysteines.     

Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma

We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody–peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody–peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody–peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed.     

On‐line biodegradation monitoring of nitrogen‐containing compounds by membrane inlet mass spectrometry

Membrane inlet mass spectrometry (MIMS) has been developed for the on‐line monitoring of compounds in a continuous stirred tank bioreactor (CSTB) used to simulate a wastewater treatment plant (WWTP). A mixture of four industrially relevant nitrogen‐containing volatile and semi‐volatile compounds was fed to a 3 dm³ CSTB with a hydraulic retention time (HRT) of 24 h. In‐membrane preconcentration/thermal desorption was used for the on‐line monitoring of semi‐volatile compounds (n‐methylpyrrolidinone and tetramethylethylenediamine), while volatile organic compounds (3‐bromopyridine and 2‐chloro‐5‐trifluoromethylaniline) were determined by continuous infusion through the membrane. Quantification of the four compounds was achieved by MS. The CSTB was run for 160 h and, after reaching steady state, n‐methylpyrrolidinone showed removal to levels below the limit of detection, while there was partial biodegradation of 2‐chloro‐5‐trifluoromethylaniline and 3‐bromopyridine. There was no evidence of significant biodegradation for tetramethylethylenediamine. Intermediates in the metabolite pathways of 2‐chloro‐5‐trifluoromethylaniline and 3‐bromopyridine were identified by MIMS, gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS). COD was measured off line, and results agreed well with MIMS, although COD data did not provide information on the removal of the individual compounds. Copyright © 2003 Society of Chemical Industry     

High‐resolution thermogravimetry/coupled mass spectrometry analysis of flame‐retardant rubber

A bromobutyl rubber composition containing a variety of conventional flame retardants, such as Saytex (decabromodiphenyl oxide), Sb₂O₃, chlorinated paraffin wax, and polychloroprene rubber, was prepared and used to coat nylon 6 fabric in a laboratory‐coating device. An attempt was made to evaluate the decomposition profile, the evolved gases, and the kinetics of the decomposition process at a dynamic heating rate with high‐resolution thermogravimetric analysis (HR‐TGA). HR‐TGA was used with mass spectrometry for evolved gas analysis (EGA). The HR‐TGA results were compared with results from conventional thermogravimetric analysis (TGA) at a constant heating rate; the former offered sharp transitions, an economic timescale, and an accurate activation energy. The resolution optimization for stability analysis and the effect of its variation on the kinetic parameters offered better results for HR‐TGA than conventional TGA. A lifetime and temperature relationship was evaluated in HR‐TGA with Toop's method, and it was observed that the shelf life decreased sharply with temperature. The effluents HBr, HCl, Br ·, and Cl ·, generated between 210 and 496°C during EGA, were correlated with the thermal stability and fire‐retardancy behavior of the material. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 2051–2057, 2003     

Matrix‐assisted laser desorption/ionization mass spectrometry of synthetic polymers. VI. Analysis of phenol–urea–formaldehyde cocondensates

Phenolic resoles can be regarded as copolymers of phenol and formaldehyde that are distributed in the chain length and the number of methylol groups per molecule. While other spectroscopic methods like FTIR and NMR only give average structures, MALDI–TOF mass spectrometry is able to resolve the oligomer distribution of phenolic resoles. Using 2,5‐dihydroxybenzoic acid or 2,4,6‐trihydroxyacetophenone as matrices, MALDI–TOF spectra are obtained where each oligomer peak can be assigned to a particular chemical structure. Thus, the degree of polymerization and the number of reactive methylol groups can be determined. For urea‐modified resoles, in addition to phenol–formaldehyde and urea–formaldehyde structures, for the first time, phenol–urea–formaldehyde cocondensate structures can be identified directly. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 2540–2548, 2003