Biomedical subjects

Daniel C Liebler

Publications and source records attributed to Daniel C Liebler.

28 records · Page 2Linked to original sources

PubMed2003

Proteomic characterization of metabolites, protein adducts, and biliary proteins in rats exposed to 1,1-dichloroethylene or diclofenac.

A proteome profiling approach was used to compare effects of two toxicants, 1,1-dicloroethylene (DCE) and diclofenac, which covalently adduct hepatic proteins. Bile was examined as a potential source of protein alterations since both toxicants target the hepatic biliary canaliculus. Bile was collected before and after toxicant treatment. Biliary proteins were separated by one-dimensional SDS-PAGE and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with data-dependent scanning. Comprehensive analysis of biliary proteins was performed by using SEQUEST and BLAST database searching, in combination with de novo interpretation. Bile not subjected to tryptic digestion was analyzed for DCE metabolites. DCE treatment resulted in a marked increase in the overall number of biliary proteins, whereas few changes in the proteomic profile were apparent in bile after diclofenac treatment. This is consonant with prior observations of more profound effects of DCE on canalicular membrane integrity. LC-MS-MS analyses for DCE metabolites revealed the presence of S-carboxymethyl glutathione, S-(cysteinylacetyl)glutathione, and a product of the intramolecular rearrangement of the DCE metabolite, ClCH(2)COSG, not previously described in vivo. In addition, several S-carboxymethylated proteins were identified in bile from DCE-treated animals. This investigation has produced the first comprehensive baseline characterization of the content of the rat biliary proteome and the first documentation of alterations in the proteome of bile by toxicant treatment. In addition, the results provide direct in vivo evidence for DCE metabolic routes proposed in the formation of covalent adducts.

Animals↗

PubMed2002

Vitamin E oxidation in human atherosclerotic lesions.

Oxidation of low-density lipoproteins (LDL) is a key process in atherogenesis, and vitamin E (alpha-tocopherol, TOH) has received attention for its potential to attenuate the disease. Despite this, the type and extent of TOH oxidation and its relationship to lipid oxidation in the vessel wall where lesions develop remain unknown. Therefore, we measured oxidized lipids, TOH, and its oxidation products, alpha-tocopherylquinone (TQ), 2,3- and 5,6-epoxy-alpha-tocopherylquinones by gas chromatography-mass spectrometry analysis in human lesions representing different stages of atherosclerosis. We also oxidized LDL in vitro to establish "footprints" of TOH oxidation product for different oxidants. The in vitro studies demonstrated that tocopherylquinone epoxides are the major products when LDL is exposed to the one-electron (ie, radical) oxidants, peroxyl radicals, and copper ions, whereas TQ preferentially accumulates with the two-electron (nonradical) oxidants, hypochlorite, and peroxynitrite. In human lesions, the relative extent of TOH oxidation was maximal early in the disease where it exceeded lipid oxidation. Independent of the disease stage, TQ was always the major oxidation product with all products together representing <20% of the total TOH present, and the oxidation product profile mirroring that formed during LDL oxidation by activated monocytes in the presence of nitrite. In contrast, oxidized lipid increased with increasing disease severity. These results suggest that two-electron oxidants are primarily responsible for TOH oxidation in the artery wall, and that the extent of TOH oxidation is limited yet substantial lipid oxidation takes place. This study may have important implications regarding antioxidant supplements aimed at preventing LDL oxidation and hence atherogenesis.

Adult↗

PubMed2002

Peptide sequence motif analysis of tandem MS data with the SALSA algorithm.

We have developed a pattern recognition algorithm called SALSA (scoring algorithm for spectral analysis) for the detection of specific features in tandem MS (MS-MS) spectra. Application of the SALSA algorithm to the detection of peptide MS-MS ion series enables identification of MS-MS spectra displaying characteristics of specific peptide sequences. SALSA analysis scores MS-MS spectra based on correspondence between theoretical ion series for peptide sequence motifs and actual MS-MS product ion series, regardless of their absolute positions on the m/z axis. Analyses of tryptic digests of bovine serum albumin (BSA) by LC-MS-MS followed by SALSA analysis detected MS-MS spectra for both unmodified and multiple modified forms of several BSA tryptic peptides. SALSA analysis of MS-MS data from mixtures of BSA and human serum albumin (HSA) tryptic digests indicated that ion series searches with BSA peptide sequence motifs identified MS-MS spectra for both BSA and closely related HSA peptides. Optimal discrimination between MS-MS spectra of variant peptide forms is achieved when the SALSA search criteria are optimized to the target peptide. Application of SALSA to LC-MS-MS proteome analysis will facilitate the characterization of modified and sequence variant proteins.

Algorithms↗

PubMed2002

Sequence mapping of epoxide adducts in human hemoglobin with LC-tandem MS and the SALSA algorithm.

The rapid development and integration of liquid chromatography-tandem mass spectrometry (LC-MS-MS) has enabled the high-throughput identification of proteins and driven the expanding field of proteomics. LC-MS-MS also offers an attractive general approach to the analysis of xenobiotic adducts on proteins. The aim of this study was to examine the combined use of LC-MS-MS and the SALSA algorithm as a general approach to map xenobiotic adducts on proteins at the level of amino acid sequence. Hemoglobin (Hb) adducts are commonly used as biomarkers for exposure to environmental toxicants. Human Hb was incubated with styrene oxide, ethylene oxide, and butadiene dioxide (40 mM) to form adducts, digested with trypsin and analyzed by LC-MS-MS on a ThermoFinnigan LCQ ion trap MS instrument. Data-dependent scanning was used for acquisition of MS-MS spectra. The SALSA algorithm was used to detect MS-MS spectra of native and modified Hb peptides. The adducted sites identified are the N-terminal valines of both Hbalpha and Hbbeta, glutamic acid 7, cysteine 93, and histidines 77, 97, and 143 of the beta chain and histidine 45 of the alpha chain. Specific shifts in the b- and y-ion series in MS-MS spectra confirmed the locations of each adduct. This approach offers a means to simultaneously identify multiple Hb adducts resulting from exposures to known or unknown toxicants. Combined application of LC-MS-MS and SALSA thus provides a general means of mapping protein modifications at the level of amino acid sequence.

Algorithms↗

PubMed2002

Alkylation and inactivation of human glutathione transferase zeta (hGSTZ1-1) by maleylacetone and fumarylacetone.

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate or maleylacetone (MA) to fumarylacetoacetate or fumarylacetone (FA), respectively. GSTZ1-1 also catalyzes the glutathione-dependent biotransformation of a range of alpha-haloacids, including dichloroacetic acid. The objective of this study was to investigate the mechanism of inactivation of hGSTZ1-1 by MA and FA and to determine the covalent modification of hGSTZ1-1 by MA and FA in the presence and absence of glutathione. MA and FA (0.01-1 mM) inactivated all hGSTZ1-1 polymorphic variants in a concentration- and time-dependent manner, and this inactivation was blocked by glutathione. The C16A mutant of hGSTZ1c-1c was partially inactivated by MA and FA. Electrospray ionization-tandem mass spectrometry and SALSA (Scoring Algorithm for Spectral Analysis) analyses of tryptic digests of hGSTZ1 polymorphic variants revealed that the active site (SSCSWR) and C-terminal (LLVLEAFQVSHPCR) cysteine residues of hGSTZ1-1 were covalently modified by MA and FA. MA and FA adduction resulted in diagnostic 156-Da shifts in the masses of the modified peptide ions and in their MS-MS fragment ions. Alkylation of the active-site cysteine residues, but not of the C-terminal cysteine, was relatively less intense when hGSTZ1-1 polymorphic variants were incubated with MA or FA in the presence of S-methyl glutathione. These data indicate that MA and FA are substrate and product inactivators of hGSTZ1-1 and covalently modify hGSTZ1-1 at the active-site cysteine residue in the absence of glutathione. The observation that inactivation was blocked by glutathione indicates that binding of glutathione to the active site prevents reaction of MA or FA with the active-site cysteine residue. These data also indicate that MA and FA may covalently modify and inactivate other proteins that have accessible cysteine residues and may, thereby, contribute to dichloroacetic acid-induced or hypertyrosinemia type-I-associated toxicities.

Acetone↗

PubMed2002

Mass spectral characterization of dichloroacetic acid-modified human glutathione transferase zeta.

Glutathione transferase zeta (GSTZ1-1) is widely expressed in eukaryotic species, and four human allelic variants of hGSTZ1-1 have been described. GSTZ1-1 catalyzes the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate and the biotransformation of a range of alpha-haloalkanoic acids. GSTZ1-1-catalyzed biotransformation of fluorine-lacking alpha,alpha-dihaloalkanoic acids, including dichloroacetic acid (DCA), results in the mechanism-based inactivation and covalent modification of the enzyme. The objective of this study was to investigate further the DCA-induced inactivation of hGSTZ1c-1c and to explore the mechanism of inactivation by characterization of the sites and types of DCA-induced covalent modifications. The partition ratio for the DCA-induced, mechanism-based inactivation of hGSTZ1c-1c was (5.7 +/- 0.5) x 10(2), and the k(cat) for the biotransformation of DCA was 39 min(-)(1). Inactivation of hGSTZ1c-1c in vitro was limited at high enzyme concentrations and was inhibited by glyoxylate. The stoichiometry of DCA binding to hGSTZ1c-1c was approximately 0.5 mol of DCA/mol of enzyme monomer. A single DCA-derived adduct was observed and was assigned to cysteine-16 by a combination of matrix-assisted laser-desorption-ionization time-of-flight and electrospray-ionization quadrupole ion-trap mass spectrometry and by analysis of [1-(14)C]DCA binding to C16A hGSTZ1c-1c. The DCA-derived adduct contained both glutathione and the carbon skeleton of DCA, presumably in a dithioacetal linkage. Also, cysteine-16 formed a mixed disulfide bond with glutathione. These data support a mechanism of inactivation whereby glutathione displaces a chlorine atom from DCA, and cysteine-16 in the enzyme active site displaces the second chlorine atom to result in a covalently modified and inactivated enzyme. These findings explain the DCA-induced inactivation of GSTZ1-1 observed in humans and rats.

Binding Sites↗

PubMed2002

Proteomic approaches to characterize protein modifications: new tools to study the effects of environmental exposures.

Proteomics is the study of proteomes, which are the collections of proteins expressed in cells. Whereas genomes are essentially invariant in different cells in an organism, proteomes vary from cell to cell, with time and as a function of environmental stimuli and stress. The integration of new mass spectrometry (MS) methods, data analysis algorithms, and information from databases of protein and gene sequences has enabled the characterization of proteomes. Many environmental agents directly or indirectly generate reactive electrophiles that covalently modify proteins. Although considerable evidence supports a key role for protein adducts in adverse effects of chemicals, limitations in analytical technology have slowed progress in this area. New applications of liquid chromatography-tandem mass spectrometry (LC-MS-MS) now offer the potential to identify protein targets of reactive electrophiles and to map adducts at the level of amino acid sequence. Use of the data-analysis tools Sequest and SALSA (Scoring Algorithm for Spectral Analysis) together with LC-MS-MS analyses of protein digests enables the identification of modified forms of proteins in a sample. These approaches can map adducts to specific amino acids in protein targets and are being adapted to searches for protein adducts in complex proteomes. These tools will facilitate the identification of new biomarkers of chemical exposure and studies of mechanisms by which protein modifications contribute to the adverse effects of environmental exposures.

Algorithms↗

PubMedDate not supplied

Quantitative analysis of modified proteins by LC-MS/MS of peptides labeled with phenyl isocyanate.

Stable isotope tagging methods have enabled relative quantitation of proteins between samples in LC-MS/MS analyses. However, most such methods are not applicable to the differential quantitation of modified proteins because the isotope tagging reagents only react with certain peptides or because the reagents incorporate a mass increment that is too small to allow reliable quantitation on low resolution ion trap MS instruments. Here, we describe the use of d0- and d5-phenyl isocyanate (PIC) as N-terminal reactive tags for essentially all peptides in proteolytic digests. PIC reacts quantitatively with peptide N-terminal amines within minutes at neutral pH and the PIC-labeled peptides undergo informative MS/MS fragmentation. Ratios of d0- and d5-PIC-labeled derivatives of several model peptides were linear across a 10000-fold range of peptide concentration ratios, thus indicating a wide dynamic range for quantitation. Application of PIC labeling enabled relative quantitation of several styrene oxide adducts of human hemoglobin in LC-MS/MS analyses. PIC labeling offers a versatile means of quantifying changes in modified or variant protein forms in paired samples.

Chemistry Techniques, Analytical↗

PubMedDate not supplied

P-Mod: an algorithm and software to map modifications to peptide sequences using tandem MS data.

The discovery of unanticipated protein modifications is one of the most challenging problems in proteomics. Whereas widely used algorithms such as Sequest and Mascot enable mapping of modifications when the mass and amino acid specificity are known, unexpected modifications cannot be identified with these tools. We have developed an algorithm and software called P-Mod, which enables discovery and sequence mapping of modifications to target proteins known to be represented in the analysis or identified by Sequest. P-Mod matches MS/MS spectra to peptide sequences in a search list. For spectra of modified peptides, P-Mod calculates mass differences between search peptide sequences and MS/MS precursors and localizes the mass shift to a sequence position in the peptide. Because modifications are detected as mass shifts, P-Mod does not require the user to guess at masses or sequence locations of modifications. P-Mod uses extreme value statistics to assign p value estimates to sequence-to-spectrum matches. The reported p values are scaled to account for the number of comparisons, so that error rates do not increase with the expanded search lists that result from incorporating potential peptide modifications. Combination of P-Mod searches from multiple LC-MS/MS analyses and multiple samples revealed previously unreported BSA modifications, including a novel decarboxymethylation or D-->G substitution at position 579 of the protein. P-Mod can serve a unique role in the identification of protein modifications both from exogenous and endogenous sources and may be useful for identifying modified protein forms as biomarkers for toxicity and disease processes.

Algorithms↗

PubMedDate not supplied

Identification of protein fragments as pattern features in MALDI-MS analyses of serum.

The use of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to acquire spectral profiles has become a common approach to detect proteomic biomarkers of disease. MALDI-MS signals may represent both intact proteins as well as proteolysis products. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis can tentatively identify the corresponding proteins Here, we describe the application of a data analysis utility called FragMint, which combines MALDI-MS spectral data with LC-MS/MS based protein identifications to generate candidate protein fragments consistent with both types of data. This approach was used to identify protein fragments corresponding to spectral signals in MALDI-MS analyses of unfractionated human serum. The serum also was analyzed by one-dimensional SDS-PAGE and bands corresponding to the MALDI-MS signal masses were excised and subjected to in-gel digestion and LC-MS/MS analysis. Database searches mapped all of the identified peptides to abundant blood proteins larger than the observed MALDI-MS signals. FragMint identified fragments of these proteins that contained the MS/MS identified sequences and were consistent with the observed MALDI-MS signals. This approach should be generally applicable to identify protein species corresponding to MALDI-MS signals.

Algorithms↗