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1.
Rapid Commun Mass Spectrom ; 34(12): e8792, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32208529

RESUMO

RATIONALE: Liquid chromatography/mass spectrometry is an essential tool for efficient and reliable quantitative and qualitative analysis and underpins much of contemporary drug metabolism and pharmacokinetics. Data-independent acquisition methods such as MSE have reduced the potential to miss metabolites, but do not formally generate quadrupole-resolved product ion spectra. The addition of ion mobility separation to these approaches, for example, in High-Definition MSE (HDMSE ) has the potential to reduce the time needed to set up an experiment and maximize the chance that all metabolites present can be resolved and characterized. We compared High-Definition Data-Dependent Acquisition (HD-DDA), MSE and HDMSE approaches using automated software processing with Mass-MetaSite and WebMetabase. METHODS: Metabolite identification was performed on incubations of glucagon-like peptide-1 (7-37) (GLP-1) and verapamil hydrochloride. The HD-DDA, MSE and HDMSE experiments were conducted on a Waters ACQUITY UPLC I-Class LC system with a VION IMS quadrupole time-of-flight (QTOF) mass spectrometer operating under UNIFI control. All acquired data were processed using MassMetaSite able to read data from UNIFI 1.9.4. WebMetabase was used to review the detected chromatographic peaks and the spectral data interpretations. RESULTS: A comparison of outcomes obtained for MSE and HDMSE data demonstrated that the same structures were proposed for metabolites of both verapamil and GLP-1. The ratio of structurally matched to mismatched product ions found by MassMetaSite was slightly greater for HDMSE than for MSE , and HD-DDA, thus improving confidence in the structures proposed through the addition of ion mobility based data acquisitions. CONCLUSIONS: HDMSE data acquisition is an effective approach for the elucidation of metabolite structures for both small molecules and peptides, with excellent accuracy and quality, requiring minimal tailoring for the compound under investigation.


Assuntos
Íons/análise , Espectrometria de Massas/métodos , Software , Cromatografia Líquida de Alta Pressão/métodos , Íons/química , Peptídeos/análise , Peptídeos/química
2.
J Biol Chem ; 292(12): 5110-5122, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28087695

RESUMO

This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin. HepG2 cells stably transfected with SMS1 (HepG2-SMS1) exhibit elevated enzyme activity in vitro and increased sphingomyelin content (mainly C22:0- and C24:0-sphingomyelin) but lower hexosylceramide (Hex-Cer) levels. HepG2-SMS1 cells have fewer triacylglycerols than controls but similar diacylglycerol acyltransferase activity, triacylglycerol secretion, and mitochondrial function. Treatment with 1 mm palmitate increases de novo ceramide synthesis in both cell lines to a similar degree, causing accumulation of C16:0-ceramide (and some C18:0-, C20:0-, and C22:0-ceramides) as well as C16:0- and C18:0-Hex-Cers. In these experiments, the palmitic acid is delivered as a complex with delipidated BSA (2:1, mol/mol) and does not induce significant lipotoxicity. Based on precursor labeling, the flux through SM synthase also increases, which is exacerbated in HepG2-SMS1 cells. In contrast, palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former. Furthermore, triacylglycerols from HepG2-SMS1 cells are enriched in polyunsaturated fatty acids, which is indicative of active remodeling. Together, these results delineate novel metabolic interactions between glycerolipids and sphingolipids.


Assuntos
Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Ceramidas/metabolismo , Diglicerídeos/metabolismo , Ácidos Graxos/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Transferases (Outros Grupos de Fosfato Substituídos)/análise , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Triglicerídeos/metabolismo , Regulação para Cima
3.
Rapid Commun Mass Spectrom ; 31(7): 591-605, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-28072489

RESUMO

RATIONALE: Refined cottonseed oil has widespread applications in the food and chemical industries. Although the major lipids comprising cottonseed oil (triacylglycerols) are well known, there are many diverse lipid species in cotton seeds that occur at much lower levels and have important nutritional or anti-nutritional properties. METHODS: The lipid technical samples were prepared in chloroform. The biological samples were extracted using a mixture of isopropanol/chloroform/H2 O (2:1:0.45). The data were collected using high and low collision energy with simultaneous data collection on a time-of-flight (TOF) mass spectrometer which allowed the characterization of lipids by precursor and product ion alignment. The supercritical fluid chromatography methodology is flexible and can be altered to provide greater retention and separation. The comprehensive method was used to screen seed lipid extracts from several cotton genotypes using multivariate statistical analysis. RESULTS: Method variables influencing the peak integrity and chromatographic separation for a mixture of lipids with different degrees of polarity were explored. The experiments were designed to understand the chromatographic behavior of lipids in a controlled setting using a variety of lipid extracts. Influences of acyl chain length and numbers of double bonds were investigated using single moiety standards. CONCLUSIONS: The methodology parameters were examined using single moiety lipid standards and standard mixtures. The method conditions were applied to biological lipid extracts, and adjustments were investigated to manipulate the chromatography. Insights from these method variable manipulations will help to frame the development of targeted lipid profiling and screening protocols. Copyright © 2017 John Wiley & Sons, Ltd.

4.
Planta Med ; 83(16): 1297-1308, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28454188

RESUMO

Ultrahigh-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UHPLC-QToF-MS) profiling was used for the identification of marker compounds and generation of metabolic patterns that could be interrogated using chemometric modeling software. UHPLC-QToF-MS was used to generate comprehensive fingerprints of three botanicals (Hoodia, Terminalia, and chamomile), each having different classes of compounds. Detection of a broad range of ions was carried out in full scan mode in both positive and negative modes over the range m/z 100-1700 using high-resolution mass spectrometry. Multivariate statistical analysis was used to extract relevant chemical information from the data to easily differentiate between Terminalia species, chamomile varieties, and quality control of Hoodia products. Using nontargeted analysis, identification of 37 compounds contributed to the differences between Terminalia species, 26 flavonoids were identified to show the differences between German and Roman chamomile, and 43 pregnane glycosides were identified from Hoodia gordonii samples. The UHPLC-QToF-MS-based chemical fingerprinting with principal component analysis was able to correctly distinguish botanicals and their commercial products. This work can be used as a basis to assure the quality of botanicals and commercial products.


Assuntos
Camomila/metabolismo , Hoodia/metabolismo , Preparações de Plantas/normas , Terminalia/metabolismo , Camomila/química , Cromatografia Líquida de Alta Pressão , Suplementos Nutricionais/normas , Hoodia/química , Espectrometria de Massas , Metaboloma , Metabolômica , Preparações de Plantas/química , Controle de Qualidade , Terminalia/química
5.
Bioanalysis ; 2022 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-35255714

RESUMO

Background: The capability of targeted MS-based methods to simultaneously measure multiple analytes with high selectivity and sensitivity greatly facilitates the discovery and quantitation of novel biomarkers. However, the complexity of biological samples is a major bottleneck that requires extensive sample preparation. Results: This paper reports a generic workflow to optimize surrogate peptide-based protein biomarker screening for seven human proteins in a multiplexed manner without the need for any specific affinity reagents. Each step of the sample processing and LC-MS methods is systematically assessed and optimized for better analytical performance. Conclusion: The established method is used for the screening of multiple myeloma patient samples to determine which proteins could be robustly measured and serve as potential biomarkers of the disease.

6.
Bioorg Med Chem Lett ; 21(1): 484-7, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21126875

RESUMO

A novel series of EP(4) ligands, based on a benzyl indoline scaffold, has been discovered. It was found that agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl group. The pharmacokinetic, metabolic and pharmacological profiles of these compounds was explored. It was found that these compounds show good pharmacokinetics in rat and are efficacious in pre-clinical models of pain and inflammation.


Assuntos
Indóis/química , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Indóis/farmacocinética , Indóis/uso terapêutico , Ligantes , Ratos , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Relação Estrutura-Atividade
7.
Bioorg Med Chem Lett ; 21(3): 1041-6, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21215624

RESUMO

Two new series of EP(4) antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP(4) receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.


Assuntos
Amidas/química , Indóis/química , Naftalenos/química , Quinolinas/química , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Compostos de Sulfonilureia/química , Amidas/síntese química , Amidas/farmacocinética , Animais , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Indóis/síntese química , Indóis/farmacocinética , Ratos , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfonilureia/síntese química , Compostos de Sulfonilureia/farmacocinética
9.
Bioanalysis ; 12(1): 53-65, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31849266

RESUMO

Aim: Developing LC-MS methods for biomolecules is often challenging due to issues with molecular size and complexity, nonspecific binding, protein binding, solubility and sensitivity. As a result, complex sample preparation workflows, including immune-affinity and/or protein digestion and lengthy analysis potentially using nano-flow LC, may be needed to achieve the required sensitivity. This work aims to provide a simple, sensitive, fast and robust method for quantification of intact IGF-I from human serum using UPLC-MS/MS. Methods: IGF-I serum samples were denatured with sodium dodecyl sulfate, followed by organic protein precipitation to effectively disrupt protein binding and subsequent SPE of the resulting supernatant for sample cleanup and enrichment prior to LC-MS/MS analysis. Separation was performed on an analytical scale LC using a reversed-phase column containing <2 µm solid core particle followed by detection on a tandem quadrupole MS in multiple reaction monitoring mode. Results: Intact IGF-I was quantified from serum using the method described above at a LLOQ of 5 ng/ml with a dynamic range 5-1000 ng/ml (r2>0.99) and mean accuracy of 101.76%. Accuracies for quality control samples were between 93.9-107.7% with RSD <7%. Conclusion: The analytical sensitivity, linear dynamic range and excellent reproducibility of this method reliably measures endogenous and elevated serum IGF-I levels, demonstrating its utility in discovery, bioanalysis and clinical research.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fator de Crescimento Insulin-Like I/análise , Espectrometria de Massas em Tandem/métodos , Biomarcadores/sangue , Humanos , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/isolamento & purificação , Limite de Detecção , Razão Sinal-Ruído , Dodecilsulfato de Sódio/química , Extração em Fase Sólida
10.
Bioanalysis ; 12(19): 1389-1403, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32975433

RESUMO

Background: Antibody biotherapeutic measurement from pharmacokinetic studies has not been traditionally based on intact molecular mass as is the case for small molecules. However, recent advancements in protein capture and mass spectrometer technology have enabled intact mass detection and quantitation for dosed biotherapeutics. A bioanalytical method validation is part of the regulatory requirement for sample analysis to determine drug concentration from in-life study samples. Results/methodology: Here, an intact protein LC-MS assay is subjected to mock bioanalytical method validation, and unknown samples are compared between intact protein LC-MS and established bioanalytical assay formats: Ligand-binding assay and peptide LC-MS/MS. Discussion/conclusion: Results are presented from the intact and traditional bioanalytical method evaluations, where the in-life sample concentrations were comparable across method types with associated data analyses presented. Furthermore, for intact protein LC-MS, modification monitoring and evaluation of data processing parameters is demonstrated.


Assuntos
Anticorpos Monoclonais/farmacocinética , Terapia Biológica/métodos , Cromatografia Líquida/métodos , Preparações Farmacêuticas/análise , Espectrometria de Massas em Tandem/métodos , Humanos
11.
Bioanalysis ; 11(13): 1275-1289, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31298556

RESUMO

Aim: Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. Results: The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4 kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. Conclusion: HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Hormônios Peptídicos/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/normas , Limite de Detecção , Hormônios Peptídicos/isolamento & purificação , Hormônios Peptídicos/normas , Controle de Qualidade , Reprodutibilidade dos Testes , Extração em Fase Sólida , Espectrometria de Massas em Tandem/normas
12.
Bioorg Med Chem Lett ; 18(6): 2048-54, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18291643

RESUMO

A new series of EP(4) antagonists based on a quinoline acylsulfonamide scaffold have been identified as part of our on-going efforts to develop treatments for chronic inflammation. These compounds show subnanomolar intrinsic binding potency towards the EP(4) receptor, and excellent selectivity towards other prostanoid receptors. Acceptable pharmacokinetic profiles have also been demonstrated across a series of preclinical species.


Assuntos
Artrite Experimental/tratamento farmacológico , Quinolinas/química , Quinolinas/farmacologia , Receptores de Prostaglandina E/antagonistas & inibidores , Receptores de Prostaglandina E/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Animais , Artrite Experimental/induzido quimicamente , Cães , Cobaias , Humanos , Macaca mulatta , Estrutura Molecular , Quinolinas/farmacocinética , Ratos , Receptores de Prostaglandina E Subtipo EP4 , Relação Estrutura-Atividade , Sulfonamidas/farmacocinética
13.
J Vis Exp ; (134)2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29733313

RESUMO

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 µg of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.


Assuntos
Cromatografia Líquida/métodos , Fragmentos de Peptídeos/análise , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Biofarmácia/métodos , Infliximab/análise , Infliximab/química , Fosforilase b/análise , Fosforilase b/química , Coelhos , Padrões de Referência , Espectrometria de Massas em Tandem/métodos
14.
Bioanalysis ; 7(11): 1397-411, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26110713

RESUMO

Capillary LC (cLC) coupled to MS has the potential to improve detection limits, address limited sample volumes and allow multiple analyses from one sample. This is particularly attractive in areas where ultrahigh assay sensitivity, low limits of detection and small sample volumes are becoming commonplace. However, implementation of cLC-MS in the bioanalytical-drug metabolism area had been hampered by the lack of commercial instrumentation and the need for experts to operate the system. Recent advances in microfabricated devices such as chip-cube and ion-key technologies offer the potential for true implementation of cLC in the modern laboratory including the benefits of the combination of this type of separation with high-resolution MS.


Assuntos
Cromatografia Líquida/instrumentação , Metabolômica/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Preparações Farmacêuticas/metabolismo , Farmacocinética , Animais , Líquidos Corporais/química , Líquidos Corporais/metabolismo , Cromatografia Líquida/métodos , Desenho de Equipamento , Humanos , Metabolômica/métodos , Camundongos , Técnicas Analíticas Microfluídicas/métodos , Preparações Farmacêuticas/sangue
15.
Bioanalysis ; 4(11): 1299-309, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22720649

RESUMO

BACKGROUND: To further expand the use of fraction collection (FC)-microplate scintillation counting (MSC) in detecting trace amount of radioactivity in absorption, distribution, metabolism and excretion (ADME) studies and improve the resolution of UHPLC-FC-MSC, we report the coupling of UHPLC with MS and faster FC (1.2 s/fraction) followed by MSC using 384-deep-well LumaPlate™ (PerkinElmer, MA, USA) for profiling of radiolabeled metabolites in plasma, urine, bile and feces. RESULTS: Collection of 1.2 s/well clearly improved the resolution of the reconstructed radiochromatograms and, at the same time, provided sufficient detection sensitivity that allowed for more accurate integration of peaks, which is required for radiolabeled ADME studies. The introduction of a reversed gradient as a make-up solvent mixture ensured more uniform drops collected in each well, with resolution maintained throughout the UHPLC run. Less sample injection and more frequent FC resulted in less quenching by matrix and accurate integration of peak. CONCLUSION: UHPLC-FC-MSC-MS is suitable for metabolite profiling in ADME studies and offers higher resolution, higher sensitivity, shorter LC running time, reduced matrix effect and more environmentally friendly experiments compared with conventional online flow scintillation analysis.


Assuntos
Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Metaboloma , Contagem de Cintilação , Animais , Radioisótopos de Carbono/química , Feminino , Química Verde , Marcação por Isótopo , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/sangue , Preparações Farmacêuticas/urina , Ratos
16.
J Med Chem ; 53(5): 2227-38, 2010 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-20163116

RESUMO

The discovery of highly potent and selective second generation EP(4) antagonist MK-2894 (34d) is discussed. This compound exhibits favorable pharmacokinetic profile in a number of preclinical species and potent anti-inflammatory activity in several animal models of pain/inflammation. It also shows favorable GI tolerability profile in rats when compared to traditional NSAID indomethacin.


Assuntos
Analgésicos/síntese química , Benzoatos/síntese química , Ciclopropanos/síntese química , Antagonistas de Prostaglandina/síntese química , Receptores de Prostaglandina E/metabolismo , Tiofenos/síntese química , Analgésicos/química , Analgésicos/farmacocinética , Animais , Benzoatos/química , Benzoatos/farmacocinética , Ciclopropanos/química , Ciclopropanos/farmacocinética , Meia-Vida , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Dor/tratamento farmacológico , Antagonistas de Prostaglandina/química , Antagonistas de Prostaglandina/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E/antagonistas & inibidores , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacocinética
17.
Rapid Commun Mass Spectrom ; 21(9): 1485-96, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17394128

RESUMO

Metabolite identification studies involve the detection and structural characterization of the biotransformation products of drug candidates. These experiments are necessary throughout the drug discovery and development process. The use of high-resolution chromatography and high-resolution mass spectrometry together with data processing using mass defect filtering is described for in vitro and in vivo metabolite identification studies. Data collection was done using UPLC coupled with an orthogonal hybrid quadrupole time-of-flight mass spectrometer. This experimental approach enabled the use of MS(E) data collection (where E represents collision energy) which has previously been shown to be a powerful approach for metabolite identification studies. Post-acquisition processing with a prototype mass defect filtering program was used to eliminate endogenous interferences in the study samples, greatly enhancing the discovery of metabolites. The ease of this approach is illustrated by results showing the detection and structural characterization of metabolites in plasma from a preclinical rat pharmacokinetic study.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Inibidores da Protease de HIV/farmacocinética , Indinavir/farmacocinética , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Biotransformação , Inibidores da Protease de HIV/sangue , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Indinavir/sangue , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley
18.
Rapid Commun Mass Spectrom ; 19(18): 2597-602, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16106348

RESUMO

The removal of bottlenecks in discovery stage metabolite identification studies is an ongoing challenge for the pharmaceutical industry. We describe the use of an 'All-in-One' approach to metabolite characterization that leverages the fast scanning and high mass accuracy of hybrid quadrupole time-of-flight mass spectrometry (QqToFMS) instruments. Full-scan MS and MS/MS data is acquired using collision energy switching without the preselection, either manually or in a data-dependent manner, of precursor ions. The acquisition of 'clean' MS/MS data is assisted by the use of ultrahigh-performance chromatography. Data acquired using this method can then be mined post-acquisition in a number of ways. These include using narrow window extracted ion chromatograms (nwXICs) for expected biotransformations, XICs for the product ions of the parent compound and/or expected modification of these product ions, and neutral loss chromatograms. This approach has the potential to be truly comprehensive for the determination of in vitro biotransformations in a drug discovery environment.


Assuntos
Espectrometria de Massas/métodos , Verapamil/análise , Verapamil/metabolismo , Animais , Cromatografia Líquida , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos , Verapamil/química
19.
Biochemistry ; 43(29): 9361-71, 2004 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-15260479

RESUMO

The solution NMR structure of a 22-residue Zn(2+)-binding domain (ZBD) from Esherichia coli preprotein translocase subunit SecA is presented. In conjunction with X-ray absorption analysis, the NMR structure shows that three cysteines and a histidine in the sequence CXCXSGX(8)CH assume a tetrahedral arrangement around the Zn(2+) atom, with an average Zn(2+)-S bond distance of 2.30 A and a Zn(2+)-N bond distance of 2.03 A. The NMR structure shows that ND1 of His20 binds to the Zn(2+) atom. The ND1-Zn(2+) bond is somewhat strained: it makes an angle of approximately 17 degrees with the plane of the ring, and it also shows a significant "in-plane" distortion of 13 degrees. A comprehensive sequence alignment of the SecA-ZBD from many different organisms shows that, along with the four Zn(2+) ligands, there is a serine residue (Ser12) that is completely conserved. The NMR structure indicates that the side chain of this serine residue forms a strong hydrogen bond with the thiolate of the third cysteine residue (Cys19); therefore, the conserved serine appears to have a critical role in the structure. SecB, an export-specific chaperone, is the only known binding partner for the SecA-ZBD. A phylogenetic analysis using 86 microbial genomes shows that 59 of the organisms carry SecA with a ZBD, but only 31 of these organisms also possess a gene for SecB, indicating that there may be uncharacterized binding partners for the SecA-ZBD.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Zinco/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Microanálise por Sonda Eletrônica , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Filogenia , Conformação Proteica , Canais de Translocação SEC , Proteínas SecA , Homologia de Sequência de Aminoácidos
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