RESUMO
Drug-induced toxicity has, in many cases, been linked to oxidative metabolism resulting in the formation of reactive metabolites and subsequent covalent binding to biomolecules. Two structurally related antipsychotic drugs, clozapine (CLZ) and olanzapine (OLZ), are known to form similar nitrenium ion reactive metabolites. CLZ-derived reactive metabolites have been linked to agranulocytosis and hepatotoxicity. We have studied the oxidative metabolism of CLZ and OLZ as well as two known metabolites of CLZ, desmethyl-CLZ (DCLZ), and CLZ-N-oxide (CLZ-NO), using in vitro rat liver microsomal (RLM) incubations with glutathione (GSH) trapping of reactive metabolites and liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Reactive metabolite binding to selected standard peptides and recombinant purified human proteins was also evaluated. Bottom-up proteomics was performed using two complementary proteases, prefractionation of peptides followed by LC-HRMS/MS for elucidating modifications of target proteins. Induced RLM was selected to form reactive metabolites enzymatically to assess the complex profile of reactive metabolite structures and their binding potential to standard human proteins. Multiple oxidative metabolites and several different GSH adducts were found for CLZ and OLZ. Modification sites were characterized on human glutathione S-transferase (hGST) alpha 1 (OLZ-modified at Cys112), hGST mu 2 (OLZ at Cys115), and hGST pi (CLZ, DCLZ, CLZ-NO and OLZ at Cys170), human microsomal GST 1 (hMGST1, CLZ and OLZ at Cys50), and human serum albumin (hSA, CLZ at Cys34). Furthermore, two modified rat proteins, microsomal GST 1 (CLZ and OLZ at Cys50) and one CYP (OLZ-modified, multiple possible isoforms), from RLM background were also characterized. In addition, direct effects of the reactive metabolite modifications on proteins were observed, including differences in protease cleavage specificity, chromatographic behavior, and charge-state distributions.
Assuntos
Clozapina/metabolismo , Glutationa Transferase/metabolismo , Olanzapina/metabolismo , Peptídeos/metabolismo , Albumina Sérica Humana/metabolismo , Cromatografia Líquida , Clozapina/química , Glutationa Transferase/química , Humanos , Estrutura Molecular , Olanzapina/química , Peptídeos/química , Ligação Proteica , Proteômica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Albumina Sérica Humana/química , Espectrometria de Massas em TandemRESUMO
RATIONALE: Acetaminophen (APAP) is a well-known analgesic, deemed a very safe over-the-counter medication. However, it is also the main cause of acute liver failure (ALF) in the Western world, via the formation of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), and its covalent attachment to liver proteins. The aim of this study was to develop a sensitive and robust quantitative assay to monitor APAP-protein binding to human serum albumin (HSA) in patient samples. METHODS: A combination of isotope dilution, peptic digestion and solid-phase extraction coupled to liquid chromatography/multiple reaction monitoring (LC/MRM) was employed. An external calibration curve with surrogate modified protein spiked into blank serum was used for absolute quantitation. Samples were analyzed by LC/MRM to measure the modified active site peptide of HSA. The LC/MRM assay was validated and successfully applied to serum samples from patients suffering from APAP-induced ALF. RESULTS: Accuracy ranged from 83.8-113.3%, within-run coefficient of variation (CV) ranged from 0.3-6.9%, and total CVs from 1.6-10.6%. Patient samples ranged from 0.12-3.91 nmol/mL NAPQI-HSA; in-between the assay dynamic range of 0.11-50.13 nmol/mL serum. In vivo median concentrations were found to be 0.62 nmol/mL and 0.91 nmol/mL for non-spontaneous survivors (n = 25) and individuals with irreversible liver damage (n = 10), respectively (p-value = 0.028), demonstrating significant potential as a biomarker for ALF outcome. CONCLUSIONS: A fast and sensitive assay was developed to accurately quantify NAPQI-HSA as a biomarker for APAP-related covalent binding in human serum.
Assuntos
Acetaminofen/efeitos adversos , Cromatografia Líquida/métodos , Falência Hepática Aguda/sangue , Albumina Sérica Humana/análise , Espectrometria de Massas em Tandem/métodos , Acetaminofen/administração & dosagem , Adulto , Estudos de Coortes , Feminino , Humanos , Falência Hepática Aguda/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Albumina Sérica Humana/metabolismoRESUMO
RATIONALE: 4-Hydroxynonenal (HNE), endogenously generated through peroxidation and breakdown of polyunsaturated fatty acids, has been linked to a number of adverse biological effects through carbonylation of essential biomolecules. Covalent binding of HNE to proteins can alter their structure and functions, causing cell damage as well as adverse immune responses. The liver plays a predominant role in metabolic transformations and hepatic proteins are often targeted by reactive metabolites. METHODS: Rat, mouse and human liver microsomes were incubated with HNE, enzymatically digested, and subjected to strong cation-exchange peptide fractionation prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis coupled to electrospray ionization quadrupole time-of-flight (QqTOF) mass spectrometry. HNE-modified peptides were detected by probability-driven peptide spectral matching and comparative analysis between treated and control samples, and confirmed based on accurate mass and high-resolution MS/MS spectra. RESULTS: A total of 99, 123 and 51 HNE-modified peptides were identified in rat, mouse and human liver microsomes related to 76, 103 and 44 target proteins, respectively. Eight proteins were found to be adducted by HNE in all three species, including ATP synthase, carbamoyl phosphate synthase, cytochrome P450 1A2, glutamate dehydrogenase 1, protein ERGIC-53, protein disulfide-isomerase, and voltage-dependent anion-selective channel protein 1. These proteins play crucial roles in cellular processes and their covalent modification could potentially alter their function and lead to cytotoxicity. CONCLUSIONS: An analytical approach was developed for the identification of in vitro HNE protein targets in rat, mouse and human liver microsomes using two-dimensional (2D) LC/MS/MS. This approach can be applied to study HNE modification of proteins in vitro and in vivo, providing insight into the toxicology of HNE protein adduction. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Aldeídos/análise , Proteínas/química , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida , Humanos , Camundongos , Microssomos Hepáticos , RatosRESUMO
RATIONALE: Isobaric interferences in human serum can potentially influence the measured concentration levels of 25-hydroxyvitamin D [25(OH)D], when low resolving power liquid chromatography/tandem mass spectrometry (LC/MS/MS) instruments and non-specific MS/MS product ions are employed for analysis. In this study, we provide a detailed characterization of these interferences and a technical solution to reduce the associated systematic errors. METHODS: Detailed electrospray ionization Fourier transform ion cyclotron resonance (FTICR) high-resolution mass spectrometry (HRMS) experiments were used to characterize co-extracted isobaric components of 25(OH)D from human serum. Differential ion mobility spectrometry (DMS), as a gas-phase ion filter, was implemented on a triple quadrupole mass spectrometer for separation of the isobars. RESULTS: HRMS revealed the presence of multiple isobaric compounds in extracts of human serum for different sample preparation methods. Several of these isobars had the potential to increase the peak areas measured for 25(OH)D on low-resolution MS instruments. A major isobaric component was identified as pentaerythritol oleate, a technical lubricant, which was probably an artifact from the analytical instrumentation. DMS was able to remove several of these isobars prior to MS/MS, when implemented on the low-resolution triple quadrupole mass spectrometer. CONCLUSIONS: It was shown in this proof-of-concept study that DMS-MS has the potential to significantly decrease systematic errors, and thus improve accuracy of vitamin D measurements using LC/MS/MS.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Vitamina D/análogos & derivados , Análise de Fourier , Humanos , Masculino , Espectrometria de Massas por Ionização por Electrospray/métodos , Vitamina D/sangue , Vitamina D/químicaRESUMO
RATIONALE: 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene derivatives (BODIPYs) are fluorescent organic dyes that are widely used as non-radioactive labels in biological analyses. The fragmentation behaviour of ten structurally related BODIPYs was studied using tandem mass spectrometry (MS/MS), to support the structural elucidation process during synthesis. METHODS: The BODIPYs were investigated by electrospray ionization (ESI)-MS/MS, utilizing collision-induced dissociation (CID) data from triple quadrupole MS and high-resolution, accurate mass CID data from Fourier transform ion cyclotron resonance (FTICR) experiments. RESULTS: Unusual radical molecular cations ([M](+â¢)) were formed directly during the ESI process. These radical species dissociated into a large range of product ions during the subsequent CID experiments. Superimposed dissociations originating from parallel [M](+â¢) and [M+H](+) decompositions significantly complicated the interpretation of the MS/MS spectra. CONCLUSIONS: Detailed dissociation mechanisms were proposed in this study for BODIPY dyes. The elemental formulae of CID product ions were unambiguously assigned using FTICR-MS and unique fragment ions were discovered for the rapid identification of methyl, ethyl, butyl, tert-butyl, and phenyl substituents of individual dyes in BODIPY synthesis mixtures by low-resolution MS.
Assuntos
Compostos de Boro/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Acetaminophen (APAP) is a mild analgesic and antipyretic used commonly worldwide. Although considered a safe and effective over-the-counter medication, it is also the leading cause of drug-induced acute liver failure. Its hepatotoxicity has been linked to the covalent binding of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), to proteins. The aim of this study was to identify APAP-protein targets in both rat and mouse liver, and to compare the results from both species, using bottom-up proteomics with data-dependent high resolution mass spectrometry and targeted multiple reaction monitoring (MRM) experiments. Livers from rats and mice, treated with APAP, were homogenized and digested by trypsin. Digests were then fractionated by mixed-mode solid-phase extraction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Targeted LC-MRM assays were optimized based on high-resolution MS/MS data from information-dependent acquisition (IDA) using control liver homogenates treated with a custom alkylating reagent yielding an isomeric modification to APAP on cysteine residues, to build a modified peptide database. A list of putative in vivo targets of APAP were screened from data-dependent high-resolution MS/MS analyses of liver digests, previous in vitro studies, as well as selected proteins from the target protein database (TPDB), an online resource compiling previous reports of APAP targets. Multiple protein targets in each species were found, while confirming modification sites. Several proteins were modified in both species, including ATP-citrate synthase, betaine-homocysteine S-methyltransferase 1, cytochrome P450 2C6/29, mitochondrial glutamine amidotransferase-like protein/ES1 protein homolog, glutamine synthetase, microsomal glutathione S-transferase 1, mitochondrial-processing peptidase, methanethiol oxidase, protein/nucleic acid deglycase DJ-1, triosephosphate isomerase and thioredoxin. The targeted method afforded better reproducibility for analysing these low-abundant modified peptides in highly complex samples compared to traditional data-dependent experiments.
RESUMO
The α,ß-unsaturated aldehyde 4-hydroxynonenal (HNE) is formed through lipid peroxidation during oxidative stress. As a highly reactive electrophile, it is able to form adducts with various biomolecules, including proteins. These protein modifications could modulate many signaling pathways, as well as cell differentiation and proliferation, and thus could be highly important in the context of the extracellular matrix and degradation of articular cartilage. This study specifically investigated the role of HNE as a bioactive molecule in chondrocytes of osteoarthritis (OA) patients. Chondrocyte extracts of OA and non-OA patients were analyzed for HNE binding using Western blot and bottom-up LC-MS/MS analyses. HNE-modified histones, H2A and H2B, and histone deacetylase were identified using anti-HNE antibodies. Furthermore, peptide sequencing and database searching revealed 95 distinct HNE-modified proteins and their exact modification sites, with 88 protein adducts being unique to OA chondrocytes. HNE-proteins of specific interest included histone H2A, H2B and H4, collagen alpha-3(VI) chain, eukaryotic initiation factor 4A-I, and nucleolar RNA helicase 2. Comparing their MS/MS spectra to those of HNE-modified standard peptides further validated the six HNE-proteins. SIGNIFICANCE: HNE binding to proteins has been shown to result in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in OA development. Considering the increased levels of HNE in OA cartilage, this reactive aldehyde could play a role in OA. This work represents a clinically-relevant in vivo study to demonstrate the pathophysiological role of HNE in human OA. Since HNE binding can alter protein conformation and function, it remains highly relevant to study the effects of this modification in OA.
Assuntos
Condrócitos , Espectrometria de Massas em Tandem , Aldeídos , Cromatografia Líquida , HumanosRESUMO
Acetaminophen (APAP)-related toxicity is caused by the formation of N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite able to covalently bind to protein thiols. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, using multiple reaction monitoring (MRM), was developed to measure APAP binding on selected target proteins, including glutathione S-transferases (GSTs). In vitro incubations with CYP3A4 were performed to form APAP in the presence of different proteins, including four purified GST isozymes. A custom alkylation agent was used to prepare heavy labeled modified protein containing a structural isomer of APAP on all cysteine residues for isotope dilution. APAP incubations were spiked with heavy labeled protein, digested with either trypsin or pepsin, followed by peptide fractionation by HPLC prior to LC-MRM analysis. Relative site occupancy on the protein-level was used for comparing levels of modification of different sites in target proteins, after validation of protein and peptide-level relative quantitation using human serum albumin as a model system. In total, seven modification sites were quantified, namely Cys115 and 174 in GSTM2, Cys15, 48 and 170 in GSTP1, and Cys50 in human MGST1 and rat MGST1. In addition, APAP site occupancies of three proteins from liver microsomes were also quantified by using heavily labeled microsomes spiked into APAP microsomal incubations. A novel approach employing an isotope-labeled alkylation reagent was used to determine site occupancies on multiple protein thiols.
RESUMO
Acetaminophen (APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the Western world. APAP is bioactivated to N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite, which can subsequently covalently bind to glutathione and protein thiols. In this study, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize NAPQI binding to human glutathione S-transferases (GSTs) in vitro. GSTs play a crucial role in the detoxification of reactive metabolites and therefore are interesting target proteins to study in the context of APAP covalent binding. Recombinantly-expressed and purified GSTs were used to assess NAPQI binding in vitro. APAP biotransformation to NAPQI was achieved using rat liver microsomes or human cytochrome P450 Supersomes in the presence of GSTA1, M1, M2, or P1. Resulting adducts were analyzed using bottom-up proteomics, with or without LC fractionation prior to LC-MS/MS analysis on a quadrupole-time-of-flight instrument with data-dependent acquisition (DDA). Targeted methods using multiple reaction monitoring (MRM) on a triple quadrupole platform were also developed by quantitatively labeling all available cysteine residues with a labeling reagent yielding isomerically-modified peptides following enzymatic digestion. Seven modified cysteine sites were confirmed, including Cys112 in GSTA1, Cys78 in GSTM1, Cys115 and 174 in GSTM2, as well as Cys15, 48, and 170 in GSTP1. Most modified peptides could be detected using both untargeted (DDA) and targeted (MRM) approaches, however the latter yielded better detection sensitivity with higher signal-to-noise and two sites were uniquely found by MRM.
RESUMO
We describe a systematic comparison of high and low resolution LC-MS/MS assays for quantification of 25-hydroxyvitamin D3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupole-quadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease. Graphical Abstract á .
Assuntos
Cromatografia Líquida , Espectrometria de Massas em Tandem , Vitamina D/análogos & derivados , Humanos , Espectrometria de Massas por Ionização por Electrospray , Vitamina D/sangueRESUMO
Cyclodextrins (CDs) are a group of cyclic oligosaccharides, which readily form inclusion complexes with hydrophobic compounds to increase bioavailability, thus making CDs ideal drug excipients. Recent studies have also shown that CDs exhibit a wide range of protective effects, preventing proteins from aggregation, degradation, and folding. These effects strongly depend on the binding sites on the protein surface. CDs only exhibit weak interactions with amino acids, however; conventional analytical techniques therefore usually fail to reveal the exact location of the binding sites. Moreover, some studies even suggest that CD inclusion complexes are merely electrostatic adducts. Here, electron capture dissociation (ECD) was applied in this proof-of-concept study to examine the exact nature of the CD/peptide complexes, and CD binding sites were unambiguously located for the first time via Fourier-transform ion cyclotron resonance (FTICR) tandem mass spectrometry.
Assuntos
Sítios de Ligação , Peptídeos/química , Espectrometria de Massas em Tandem/métodos , beta-Ciclodextrinas/química , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Peptídeos/metabolismo , beta-Ciclodextrinas/metabolismoRESUMO
This short application note describes a simple and automated assay for determination of 25-hydroxyvitamin D (25(OH)D) levels in very small volumes of human serum. It utilizes commercial 96-well micro-extraction plates with commercial 25(OH)D isotope calibration and quality control kits. Separation was achieved using a pentafluorophenyl liquid chromatography column followed by multiple reaction monitoring-based quantification on an electrospray triple quadrupole mass spectrometer. Emphasis was placed on providing a simple assay that can be rapidly established in non-specialized laboratories within days, without the need for laborious and time consuming sample preparation steps, advanced calibration or data acquisition routines. The analytical figures of merit obtained from this assay compared well to established assays. To demonstrate the applicability, the assay was applied to analysis of serum samples from patients with chronic liver diseases and compared to results from a routine clinical immunoassay.