Mass spectrometric mapping of disulfide bonds in recombinant human interleukin-13

Interleukin 13 (IL-13), a member of the a-helical family of cytokines, has approximately 30% primary sequence homology with IL-4 and shares a common receptor component. The biologically active rhIL-13 is monomeric and non-glycosylated, and contains two disulfide bonds as determined by comparative electrospray mass spectrometric (MS) analysis of the protein before and after reduction with dithiothreitol-dithioerythritol. A trypsin-resistant core peptide of rhIL-13 was isolated and analyzed by plasma desorption (PD) MS, identifying a disulfide-linked core peptide. Subsequent digestion of this core peptide by pepsin, followed by PDMS analysis of the resulting cystine-containing peptic fragments, provided rapid determination of the existing disulfide bonds between cysteine residues 28-56 and 44-70. This disulfide arrangement is similar to that observed for the analogous four internal cysteine residues in hIL-4. The conservation of disulfide bond arrangements between hIL-13 and hIL-4, coupled with their alpha-helical structure and sequence homologies, confirms that IL-13 and IL-4 are structural homologues. It is also consistent with their reported similarities in biological function and receptor binding kinetics.     

Mass spectrometric characterization of gelsolin isoforms

Gelsolin is the most widely expressed member of the actin capping and severing family of proteins. There are two isoforms of gelsolin: isoform 1, a secretory (plasma) protein that is 51 amino acids longer than isoform 2, a cytosolic protein, at the N‐terminus; the first 27 amino acids is a signal sequence. Both isoforms are coded by a single gene and differ as a result of alternative initiation site/splicing. The level of gelsolin in the blood and cerebrospinal fluid (CSF) is altered in many diseases including amyloidoses and other neurodegenerative disorders. Although quantitative analysis of gelsolin has been reported, lack of suitable antibodies makes it impossible to differentiate these two isoforms by immunodetection techniques and no other technique is available. Therefore, ambiguity exists whether gelsolin present in circulation is isoform 1 or also isoform 2 released from lysed cells. We report in this communication a mass spectrometric approach to identify isoform 1 of gelsolin immunopurified from human plasma and CSF. Recombinant isoform 1 was used as reference. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell – the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated. Received: 16 December 1999 / Accepted: 17 December 1999     

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell--the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated.     

Mass spectrometric fragmentations of hexafluoroacetone ketals derived from N-alkenes

The mass spectrometric fragmentation behavior of the cyclic hexafluoroacetone ketals derived from eighteen n-alkenes is discussed with emphasis on locating the original olefinic bond. The major analytically useful fragments result from loss of CF₃, loss of an alkyl side chain or loss of an aldehyde moiety from the ring. Influences of geometric isomerism on the spectra are examined by intensity ratios and ratios of competing fragmentations.     

Mass spectrometric characterization of stathmin isoforms separated by 2D PAGE

In proteome analysis, the determination of the phosphorylation status of proteins and protein isoforms, which have been separated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), is of prime importance in addition to their identification. In this study, the extent to which such information can be directly extracted from the mass spectrometric data used for identification was evaluated. By searching for metastable peaks which are characteristic for loss of phosphoric acid, the Ser-phosphorylated peptides were identified with a high success rate in reflector matrix-assisted laser desorption/ionization (MALDI) mass maps of in-gel digested proteins. Furthermore, by employing a double enzymatic strategy using trypsin and Glu-C in parallel, improved sequence coverage and additional separation of the potential phosphorylation sites of the isoforms were achieved. The precise location of the modified sites within an identified phosphopeptide was obtained by submitting the corresponding molecular ions directly to nano-electrospray tandem mass spectrometric analysis. In this way the detailed phosphorylation status of six isomers of stathmin separated by 2D PAGE was determined. Two of these six isomers were phosphorylated at all four known sites (serines 15, 24, 37 and 62) and were probably derived from the previously reported alpha and beta forms, which differ by a yet unknown modification. In addition, isomers phosphorylated at serines 15, 24 and 37, serines 24, 37 and 62, serines 24 and 37 and serine 37 only were characterized.     

Mass spectrometric characterization of different norandrosterone derivatives by low-cost mass spectrometric detectors using electron ionization and chemical ionization

The abuse of nortestosterone in sport is an important problem in doping-control analysis. In order to detect the main urinary metabolite of nortestosterone, norandrosterone (NA), sensitive and specific methodology is necessary. In this context the use of a low-cost mass spectrometric detector such as the Finnigan MAT ion-trap detector (ITD) was studied. The electron ionization (EI) and positive-ion chemical ionization (PICI) mass spectra of the methoxime-trimethylsilyl, trimethylsilyl-enol trimethylsilyl ether and pentafluoropropionic ester derivatives of NA are described. The limits of detection of these derivatives are compared with those obtained by the Hewlett-Packard mass selective detector (MSD), another low-cost mass spectrometric detector and operating only in the EI mode. For the derivatives of the reference standard of NA the ITD has in the EI mode the same limit of detection, in the range of 0.5 to 1 ng injected on the column, as the MSD. However, under these conditions the ITD provides more spectrometric information, because it gives full scan data. Moreover, with the same or even improved limits of detection the ITD can operate in the PICI mode. On the other hand, for the analysis of NA isolated from urine samples, the performance of the MSD was better than that of the ITD. The ion trapping technique is probably limited when the chemical background is high.     

Mass spectrometric characterization of poly(ethylene terephthalate-co-p-oxybenzoate)

Copolyesters containing ethylene terephthalate and p-oxybenzoate units in mole ratios of ca. 80:20, 70:30, 40:60, and 20:80, respectively, were examined by direct pyrolysis–mass spectrometry to obtain information about the thermal stabilities and the sequences distributions of the two types of units as a function of composition. Thermal stability as measured by rate of volatilization increased with increasing oxybenzoate content, as did the amount of char residue formed. The electron impact mass spectrum of the volatile fragments showed the formation of predominantly linear fragments with carboxyl and vinyl end groups. Dimer, trimer, and tetramer fragments containing either or both types of units could be identified and their relative amounts could be estimated from peak intensities. The results were consistent with those expected for random distributions of the two units, i.e., for statistical copolymers.     

Mass spectrometric characterization of substituted 2-thiazolin-4-one derivatives

Electron ionization mass spectrometry was used for the structural characterization of substituted 2-thiazolin-4-one derivatives in the gas phase. The compounds follow common fragmentation pathways, producing ions whose abundances are dependent on the chemical nature of the substituent at position 2. Collision-induced dissociation tandem mass spectrometric experiments, carried out on both molecular ions and fragment ions produced in the source, allowed the elucidation of gas-phase decompositions. The presence of tautomeric forms is suggested for some ionic species. Rapid identification of a primary or secondary amine moiety at position 2 of the thiazoline ring can be achieved by the detection of characteristic fragmentations occurring both in the ion source and under the collision-induced dissociation regime.     

Mass spectrometric characterization of peptides containing different oxidized tryptophan residues

The term reactive oxygen species refers to small molecules that can oxidize, for example, nearby proteins, especially cysteine, methionine, tryptophan, and tyrosine residues. Tryptophan oxidation is always irreversible in the cell and can yield several oxidation products, such as 5-hydroxy-tryptophan (5-HTP), oxindolylalanine (Oia), kynurenine (Kyn), and N-formyl-kynurenine (NFK). Because of the severe effects that oxidized tryptophan residues can have on proteins, there is a great need to develop generally applicable and highly sensitive techniques to identify the oxidized residue and the oxidation product. Here, the fragmentation behavior of synthetic peptides corresponding to sequences recently identified in three skeletal muscle proteins as containing oxidized tryptophan residues were studied using postsource decay and collision-induced dissociation (CID) in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry (MS) and CID in an electrospray ionization (ESI) double quadrupole TOF-MS. For each sequence, a panel of five different peptides containing Trp, 5-HTP, Kyn, NFK, or Oia residue was studied. It was always possible to identify the modified positions by the y-series and also to distinguish the different oxidation products by characteristic fragment ions in the lower mass range by tandem MS. NFK- and Kyn-containing peptides displayed an intense signal at m/z 174.1, which could be useful in identifying accordingly modified peptides by a sensitive precursor ion scan. Most importantly, it was always possible to distinguish isomeric 5-HTP and Oia residues. In ESI- and MALDI-MS/MS, this was achieved by the signal intensity ratios of two signals obtained at m/z 130.1 and 146.1. In addition, high collision energy CID in the MALDI-TOF/TOF-MS also permitted the identification of these two isomeric residues by their v- and w-ions, respectively.     

Mass spectrometric study of oligourea macrocycles and their anion binding behavior

Two series, one of tris‐urea macrocycles and another of hexakis‐urea macrocycles, are examined by (tandem) Fourier‐transform ion cyclotron resonance (FTICR) mass spectrometry with respect to their fragmentation patterns and anion binding properties. All macrocycles are based on two different building blocks, one of which is a very rigid xanthene unit and the other one is a more flexible diphenyl ether. The composition and the sequence of these units thus determine their flexibility. During the fragmentation of deprotonated oligourea macrocycles in the gas phase, one urea N? CO bond is cleaved followed by a scrambling reaction within the macrocycle structure. Consequently, fragments are observed that deviate from those that would be expected from the sequence of the subunits. Interesting anion binding properties involve the simultaneous recognition of two chloride anions by one of the hexakis‐urea macrocycles, whose flexibility allows this host to form a double‐helical structure. Flexibility also determines which of the hexameric receptors bears a high sulfate affinity. The interaction energy between some of the macrocycles and sulfate is high enough to even stabilize the intrinsically unstable sulfate dianion. Copyright © 2009 John Wiley & Sons, Ltd.     

Mass spectrometric study of methyl-substituted 4-azaphenanthrenes and their nitration products

The mass spectral behavior of five derivatives of the 4-azaphenanthrene series — 1,3-dimethyl-(I), 2,3-dimethyl-(II), 1,2,3,-trimethyl-(III), 1,2,3-trimethyl-8-nitro-(IV), and 1,3-dimethyl-6,7-dinitro-4-azaphenanthrene (V) — was studied. The stabilities of the molecular ions with respect to gragmentation (W_M) are higher by a factor of two or more for the methyl-substituted I–III than for nitro derivatives IV and V. The intensity of the [M-H]⁺ ion peak in the mass spectra of I–V does not depend on the number of methyl groups but only on their positions: the presence of a CH₃ group in the 2 position leads to an [M-H]⁺ ion that is 1.5 times more intense than when there is a methyl group in the 1 position. The molecular ions of I–V do not eliminate HCN molecules; this constitutes evidence for the absence of randomization of their methyl groups. The presence of a CH₃ substituent in the 1 or 2 position does not affect the intensity of the [M-CH₃]⁺ ion peaks, while the simultaneous presence of CH₃ groups attached to the C₁ and C₂ atoms increases the intensity of the [M-CH₃]⁺ fragment peak by a factor of two. In the mass spectra of nitro derivatives IV and V, [M-O]⁺, [M-OH]⁺, [M-NO]⁺, and [M-NO₂]⁺ fragments are observed in the first step of the fragmentation of the M⁺ ion, whereas the [M-CO]⁺ ion peak characteristic for the dissociative ionization of 1-nitronaphthalene is also observed for 8-nitro-substituted IV.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1365–1369, October, 1977.     

Mass spectrometric characterization and gas-phase chemistry of self-assembling supramolecular squares and triangles

A detailed mass spectrometric characterization of self-assembling polynuclear metal complexes is described. The complexes can only be ionized as intact species under a surprisingly narrow range of conditions by electrospray ionization. Comparison with the results from NMR experiments shows that several solution-phase features of these squares and triangles (such as trends in bond energies, ligand-exchange reactions, or square-triangle equilibria) are qualitatively reflected in the gas-phase data. Consequently, mass spectrometry represents a valuable method for the characterization of these compounds. Nevertheless, the formation of unspecific aggregates during the ionization process occurs and its implications are discussed. Beyond the chemistry in solution, the fragmentation pathways of these complexes in the gas phase have been studied by infrared multiphoton dissociation (IRMPD) experiments. The results of IRMPD studies allow us to draw conclusions with respect to the structure and energetics of fragmentation products. In this tandem MS experiment, reaction pathways can be observed directly which can hardly be analyzed in solution. According to these results, the equilibration of triangles and squares involves the supramolecular analogue of a neighboring-group effect.     

Mass spectrometric characterization of low-molecular-mass color pI markers and their use for direct determination of pI value of proteins

The use of low-molecular-mass color pI markers for the determination of pI values of proteins in gel isoelectric focusing (IEF) in combination with mass spectrometry is described. Different types of substituted phenols of known pI values within the mass range 250-400 were used here as pI markers. The pure, synthesized pI markers were studied by MALDI-TOF/TOF MS. Fragmentation studies of the pI markers were also performed. Only stable and well-characterized pI markers were used in this work. The selected pI markers were mixed with proteins, deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of the focusing process and also estimation of the position of the invisible focused bands. The separated bands of the pI markers (containing separated proteins) were excised, and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. From the washed gel pieces the remaining carrier ampholytes were then washed out and proteins were in-gel digested with trypsin. The obtained peptides were measured by MALDI-TOF/TOF MS and the proteins identified via a protein database search. This procedure allows avoiding time-consuming protein staining and destaining procedures, which shortens the analysis time roughly by half. For comparison, IEF gels were stained with Coomassie Brilliant Blue R 250 and proteins in the gel bands were identified according to the standard proteomic protocol. This work has confirmed that our approach can give information about the correct pI values of particular proteins and shorten significantly the time of analysis.     

Mass spectrometric investigation of 2-aminopropiophenones and some of their metabolites.

Complex metabolic mixtures of 2-aminopropiophenones, obtained both after in vitro and human in vivo metabolism of these compounds, have been investigated using both mass spectrometry and gas chromatography/mass spectrometry. The mass spectrometric fragmentation schemes of the compounds have been proposed and verified. The schemes are based on the characteristic fragments obtained by alpha-cleavage of these compounds using direct inlet mass spectrometry or gas chromatography/mass spectrometry. These findings were confirmed with chemical ionization mass spectrometry, when quasi-molecular (MH+) ions were obtained as the highest relative abundance ions for all the compounds investigated, and were used in metabolic investigations of 2-aminopropiophenones.     

Mass spectrometric characterization of urinary toremifene metabolites for doping control analyses

Toremifene is a selective estrogen receptor modulator included in the list of prohibited substances in sport by the World Anti-doping Agency. The aim of the present study was to investigate toremifene metabolism in humans in order to elucidate the structures of the most abundant urinary metabolites and to define the best marker to detect toremifene administration through the analysis of urine samples. Toremifene (Fareston) was administered to healthy volunteers and the urine samples were subjected to different preparation methods to detect free metabolites as well as metabolites conjugated with glucuronic acid or sulphate. Urinary extracts were analyzed by LC-MS/MS with triple quadrupole analyzer using selected reaction monitoring mode. Transitions for potential metabolites were selected by using the theoretical [M+H](+) as precursor ion and m/z 72 or m/z 58 as product ions for N,N-dimethyl and N-desmethyl metabolites, respectively. Toremifene and 20 metabolites were detected in excretion study samples, excreted free or conjugated with glucuronic acid or sulphate. Structures for most abundant phase I metabolites were proposed using accurate mass measurements performed by QTOF MS, based on fragmentation pattern observed for those metabolites available as reference standards. Several metabolic pathways including mono- and di-hydroxylation, N-desmethylation, hydroxymethylation, oxidation, dehalogenation and combinations were proposed. All metabolites were detected up to one month after toremifene administration; the most abundant metabolites were detected in the free fraction and they were metabolites resulting from dehalogenation. Several of the metabolites elucidated in this work have not been reported until now in the scientific literature.