Biomedical subjects
Publications and source records attributed to C Fenselau.
Corona plasma discharge provides a rapid and reliable tool for release of biomarkers from gram negative and positive bacteria, spores and viruses for characterization by mass spectrometry.
Unique patterns of biomarkers were reproducibly characterized by matrix-assisted laser desorption ionization (MALDI)-mass spectrometry and were used to distinguish Bacillus species members from one another. Discrimination at the strain level was demonstrated for Bacillus cereus spores. Lipophilic biomarkers were invariant in Bacillus globigii spores produced in three different media and in B. globigii spores stored for more than 30 years. The sensitivity was less than 5,000 cells deposited for analysis. Protein biomarkers were also characterized by MALDI analysis by using spores treated briefly with corona plasma discharge. Protein biomarkers were readily desorbed following this treatment. The effect of corona plasma discharge on the spores was examined.
Acquired drug resistance is one of the most important problems in cancer chemotherapy. One of the proposed mechanisms for these phenomena is the sequestration of alkylating agents by metallothionein in vivo. This research shows that metallothionein can covalently sequester phosphoramide mustard, the active form of cyclophosphamide in vitro. On-line electrospray mass spectrometry reveals that it is phosphoramide, not nornitrogen mustard that alkylates metallothionein, although the metallothionein/nornitrogen mustard adduct was isolated as the major adduct. Tandem mass spectrometric experiments were performed on an isolated drug-modified tryptic peptide. The alkylation occurred predominantly at Cys48 of metallothionein. These results provide further evidence that overexpression of metallothionein can detoxify the active form of the drugs.
Various sugars, peptides, and lipids were analyzed on a Fourier transform mass spectrometer using laser desorption and ionization with and without the assistance of matrixes. A compact Nd:YAG laser with an output at 1.06 microns corresponding to fundamental frequency was employed. Gram-negative and Gram-positive bacteria were also subjected to laser desorption mass spectrometry. Characteristics ions of conjugated lipid, formed by attachment of alkali metal cations, endogenous to the cells, were observed. Particle/liquid matrixes (e.g., cobalt in glycerol) proved to be useful with the 1.06-micron laser. The particles absorb efficiently laser radiation in a broad wavelength range. The liquid provides the same advantages as in fast atom bombardment: increased signal-to-noise ratios and enhanced sample lifetimes. The effect of laser power on total ion current was shown to differ for samples with and without the particle/liquid matrix. The Fourier transform analyzer provides MS/MS capability for both positive and negative ions from complex mixtures. Ions desorbed externally are introduced into the cell via a quadrupole ion guide with a lower mass cutoff. Such a setup allows matrix ions to be excluded and thus provides excellent signal-to-noise ratios for lower mass range fragment ions formed inside the cell.
Matrix-assisted laser desorption/ionization mass spectrometry has enabled viral coat proteins to be characterized directly from the virus. This analysis, demonstrated here with tobacco mosaic virus U2, a bacteriophage MS2, and equine encephalitis TRD, is achieved with a combination of organic acid, UV-absorbing matrix, and high-energy desorption with a nitrogen laser. The molecular weights of these proteins are determined with sufficient accuracy to allow differentiation among viral species and strains. The abundant hydrophobic MS2 coat protein was analyzed in aliquots of culture medium and of the tobacco mosaic virus coat protein in infected leaves. This method provides rapid detection of coat protein in the low-femtomole range, as estimated by titering plaque-forming units of MS2.
The capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil-alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be determined for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc-bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single-stranded DNA.
The research reported here demonstrates covalent binding to the metal-binding protein metallothionein (MT) by the therapeutic nitrogen mustard mechlorethamine. The most surprising aspect of this interaction is the selectivity of the alkylating agent for specific residues of MT. A combination of MS and proteolytic and enzymatic methods was used to deduce specific locations of mechlorethamine alkylation. These experiments indicated that alkylation occurs predominantly in the carboxyl domain of MT, with one molecule of mechlorethamine covalently cross-linking two cysteine residues. Electrospray MS revealed the retention of all seven metal ions in the cross-linked MT/mechlorethamine adducts, highlighting the uniqueness of this protein. Computerized docking experiments supported the hypothesis that selective binding precedes selective alkylation, and the structure of the drug indicates the minimal structural requirements for this binding. These results support the idea that MT overexpressed in tumor cells contributes to the inactivation of anticancer drugs.
The stability of single beta-strands and multistrand beta-pleated sheets as elements of secondary structure is examined in the absence of intermolecular interactions. Such experimental conditions (e.g., complete removal of solvent molecules and counterions) are achieved by placing the peptide ions in the gas phase. The metastable multiply- charged peptide ions produced by electrospray ionization undergo unimolecular dissociation. Intercharge repulsion within the precursor ions gives rise to the elevated kinetic energy of fragment ions, which is measured using Mass-analyzed Ion Kinetic Energy (MIKE) spectrometry. Intercharge distances calculated based on these measurements are compared to the numbers derived from molecular mechanics calculations with charge site assignments based on relative proton affinities. Evidence is presented suggesting that single beta-strands form collapsed structures in the absence of solvents, while multistrand beta-pleated sheets are likely to retain "native-like" secondary structures under the same conditions. These results indicate that intramolecular hydrogen bonds are the major factor determining the three-dimensional arrangements of polypeptides in the gas phase, compensating both long- and short-range electrostatic repulsions. This is in good agreement with our earlier findings (Proteins 27:165170, 1997) concerning stability of helical conformation of melittin in the absence of solvent.
Explore the source record for details and available documents.
The stability of the alpha helix as an element of secondary structure is examined in the absence of solvation, in the gas phase. Mass-analyzed ion kinetic energy (MIKE) spectrometry was applied to measure intercharge repulsion and intercharge distance in multiply protonated melittin, a polypeptide known to possess a stable helical structure in a number of different environments. The experimental results, interpreted in combination with molecular mechanics calculations, suggest that triply charged melittin retains its secondary structure in the gas phase. The stability if the alpha-helical conformation of the polypeptide in the absence of solvent molecules reflects the fact that a network of intrinsic helical hydrogen bonds is energetically more favorable than unfolded conformations.
It is of interest to test the hypothesis that induced metallothionein (MT) acts in acquired drug resistance by covalent sequestration. In this study MT was incubated in vitro with chlorambucil (CHB) under conditions where only 1:1 covalent adducts were formed. The proteolytic products of these adducts were analyzed by HPLC and mass spectrometry to reveal two major sites of modification. These were the sulfur atoms of cysteines 33 and 48, which cochelate the same metal atom in native MT. The time course of the reaction was followed using on-line electrospray ionization with a double-focusing mass spectrometer. These experiments showed that drug-modified MT binds seven metal ions, as does the unmodified protein. Molecular docking experiments showed that the selectively of drug binding is influenced by the presence of the aziridinium ion in the drug structure and complementary charge densities in the protein structure.
Derivatized bovine adenosine deaminase is used in enzyme replacement therapy and as an adjunct to gene therapy against severe combined immunodeficiency syndrome. Although a gene sequence is known for human adenosine deaminase, the structure of the bovine enzyme has not been characterized. Structure studies using mass spectrometry are reported here that evaluate sequence, processing, post-translational modifications and the extent of homology between the human protein and its therapeutic surrogate.
Interaction of chlorambucil and the glutathione-depleted human placenta pi class glutathione S-transferase (pi GST) results in the formation of a complex between the drug and the protein at physiological pH. This complex is not formed in the presence of glutathione or S-hexylglutathione. Molecular mass measurement of the reaction product using matrix-assisted laser desorption mass spectrometry indicates that one molecule of chlorambucil cross-links two subunits of the homodimeric protein. A combination of enzymic proteolysis, high performance liquid chromatography, and mass spectrometry reveals that chlorambucil alkylation occurs at cysteine 47 of one subunit and cysteine 101 of the second subunit. This result supports the idea that conformational changes occur in glutathione-depleted pi GST, which allow the bifunctional tether of chlorambucil to cross-link the two subunits of the protein.
Oxidants targeted toward inactivation of the nucleocapsid zinc finger protein are under development as antiviral agents, especially for use against human immunodeficiency virus. In the present study, electrospray ionization-mass spectrometry is used to follow in situ the progress of the reactions of 2,2'-dithiodipyridine and disulfiram with recombinant nucleocapsid protein p7 (Ncp7) from human immunodeficiency virus-1 at pH 7.4. Both reagents react with the two zinc fingers in the protein, resulting in the ejection of two zinc ions and the formation of oxidized apo-Ncp7 with three intramolecular disulfide bonds. The ejection of zinc by 2,2'-dithiodipyridine occurs in two steps. Alkylation of unreacted cysteine residues with N-ethylmaleimide after a 2-min reaction with 2,2'-dithiodipyridine reveals that the carboxyl-terminal zinc finger is disrupted first. Cys-49, Cys-36, and, to a lesser extent, Cys-39 are all shown to be target residues for initial electrophilic attack. In the reaction of disulfiram with Ncp7, ejection of the two zinc ions also occurs in two steps; however, the fully oxidized apo-Ncp7 is formed more rapidly. Thus, after a 40-min reaction, 45% of native Ncp7 is oxidized by 2,2'-dithiodipyridine, whereas 75% is oxidized by disulfiram.
Rabbit liver metallothionein-2 is shown to form covalent bonds with the anticancer agent melphalan, in support of the hypothesis that covalent sequestration by metallothionein constitutes one mechanism for the cross-resistance acquired by cancer patients to therapeutic alkylating agents. Among 20 cysteines in the 2-domain protein, 89% of the first alkylation reaction occurs with 2 that cochelate a zinc cation in the carboxy domain. Computer-supported docking studies indicate a favorable binding site for melphalan near these cysteine sulfhydryl groups. Although folded metallothionein-2 is resistant to trypsin cleavage, alkylation by 1 mol of melphalan allows cleavage by trypsin between the two globular domains.
Microsomal glutathione S-transferase (mGST) was purified to homogeneity from male Sprague-Dawley rat liver, as determined by SDS-PAGE. Removal of Triton X-100 and further separation by reversed phase HPLC revealed two proteins, mGST 1 and mGST 2, in a 1:3 ratio. Analysis of mGST 1 and mGST 2 by electrospray ionization mass spectrometry determined their molecular weights to be 17,354.2 +/- 6.6 and 17,397.9 +/- 6.6, respectively. mGST 1 was in close agreement with the calculated molecular weight of 17,348, as predicted by the previously reported cDNA sequence. Cyanogen bromide digestion and peptide mapping by fast atom bombardment mass spectrometry (FAB-MS) localized the mass increase to the N-terminal peptide, 1-7. FAB-tandem mass spectrometry of this peptide in conjunction with Edman reactions on the intact protein demonstrated the N-terminal alanine to be acetylated.
Electroblotting proteins separated by gel electrophoresis provides a suitable support for further manipulations and analysis of small amounts of relatively pure samples. On-membrane digestion, peptide mapping by mass spectrometry, and database searching offer sensitive and fast tools to identify the analyte. By providing sequence information, tandem mass spectrometry can go a step further, confirming the database identification, solving problems connected with post-translational modifications and sequence variations, or supplying the stretches of internal sequence necessary to synthesize an oligonucleotide probe for gene isolation. The viability of this approach was successfully evaluated using different tandem mass spectrometric techniques: metastable decomposition in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight instrument with a curved-field reflectron; low energy collision-induced dissociation in a MALDI quadrupole ion trap mass spectrometer; and high energy collision-induced dissociation in a high-performance four-sector mass spectrometer with massive cluster-impact ionization.
A microbore high pressure liquid chromatograph has been interfaced to a Vestec electrospray ionization source retrofitted to a Hewlett-Packard quadrupole mass spectrometer. The chief features include the absence of a splitter, the use of a second, ballast column to provide a stable flow rate across the gradient, and an in-line UV detector. The system was evaluated for analysis of peptide mixtures and applied to identification of drug-modified peptides released by tryptic digestion of drug-alkylated metallothionein.