RESUMO
We report here a novel approach for the extraction and analysis of thyroid hormones (TH) and their metabolites (THM) from human serum samples. Our method features a compact, 96-well micro-titre plate-based pre-analytic extraction/clean-up workflow combined with an isotope dilution LC-MS/MS-MS3 analytical method. In particular, these features make possible the detection of iodothyronines at their endogenous concentrations in serum differing by a factor of ca. 104, with potential to semi-automate the pre-analytics. The method was validated by the assessment of linearity, lower limits of quantification and detection (LLOQ and LLOD respectively), intra- and inter-day accuracy, precision, process efficiency (PE), matrix effect (ME) and relative recovery (RE). Calibration curves were linear in the concentration range in sample matrix from 0.1-250 nM for T3, rT3, T4 and 3-T1AM and from 0.005-1 nM for 3,5-T2 and 3,3'-T2. Using a 200-µL sample volume, the analyte dependant LLOQ were in the range 0.005 (3,5-T2) to 0.25 (T4) nM and LLOD were between 0.002 (3,5-T2) and 0.052 nM (T4). We applied the LC-MS/MS-MS3 method to the analysis of a cross section of patients with disorders of the thyroid hormone axis. T4, T3 and rT3 concentrations (± standard deviation) were 120 ± 18, 1.9 ± 0.4 and 0.45 ± 0.09 nM respectively. 3,3'-T2 concentrations (± standard deviation) were 0.079 ± 0.022 nM; 3,5-T2 concentrations were below the LLOQ and/or LLOD in all but a single sample (0.013 nM). This method expands the analytical spectrum to endogenous thyroid hormone metabolites such as 3,5-T2 which exert biological actions and rT3 which may act as surrogate markers for disturbed thyroid hormone metabolism. Graphical abstract.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Tironinas/sangue , Calibragem , Humanos , Limite de Detecção , Padrões de Referência , Reprodutibilidade dos Testes , Tironinas/normasRESUMO
The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate in the upper transmembrane helix 6 (Asp(6.59)) of the receptor as the first position that directly interacts with arginine 19 of the ligand (Arg(19)). Replacement of Asp(6.59) with Arg(19) of PrRP20 led to D6.59R, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics Arg(19) by interacting with additional binding partners in the receptor. Next, we generated an initial comparative model of this CAM because no ligand docking was required, and we selected the next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process, we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains, we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for prolactin-releasing peptide, rather than a deep, membrane-embedded binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCRs and might facilitate the development of GPCR ligands, but it also provides new targeting of CAMs involved in hereditary diseases.
Assuntos
Mutação , Hormônio Liberador de Prolactina/química , Prolactina/química , Receptores Acoplados a Proteínas G/química , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Clonagem Molecular , Desenho de Fármacos , Vetores Genéticos , Células HEK293 , Humanos , Concentração Inibidora 50 , Ligantes , Dados de Sequência Molecular , Mutagênese , Peptídeos/química , Ligação Proteica , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Mutagenic investigations of expressed membrane proteins are routine, but the variety of modifications is limited by the twenty canonical amino acids. We describe an easy and effective cysteine substitution mutagenesis method to modify and investigate distinct amino acids in vitro. The approach combines the substituted cysteine accessibility method (SCAM) with a functional signal transduction readout system using different thiol-specific reagents. We applied this approach to the prolactin-releasing peptide receptor (PrRPR) to facilitate biochemical structure-activity relationship studies of eight crucial positions. Especially for D(6.59)C, the treatment with the positively charged methanethiosulfonate (MTS) ethylammonium led to an induced basal activity, whereas the coupling of the negatively charged MTS ethylsulfonate nearly reconstituted full activity, obviously by mimicking the wild-type charged side chain. At E(5.26)C, W(5.28)C, Y(5.38)C, and Q(7.35)C, accessibility was observed but hindered transfer into the active receptor conformation. Accordingly, the combination of SCAM and signaling assay is feasible and can be adapted to other G-protein-coupled receptors (GPCRs). This method circumvents the laborious way of inserting non-proteinogenic amino acids to investigate activity and ligand binding, with rising numbers of MTS reagents allowing selective side chain modification. This method pinpoints to residues being accessible but also presents potential molecular positions to investigate the global conformation.
Assuntos
Cisteína/química , Receptores Acoplados a Proteínas G/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Ligação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The prolactin releasing peptide (PrRP) is involved in regulating food intake and body weight homeostasis, but molecular details on the activation of the PrRP receptor remain unclear. C-terminal segments of PrRP with 20 (PrRP20) and 13 (PrRP8-20) amino acids, respectively, have been suggested to be fully active. The data presented herein indicate this is true for the wildtype receptor only; a 5-10-fold loss of activity was found for PrRP8-20 compared to PrRP20 at two extracellular loop mutants of the receptor. To gain insight into the secondary structure of PrRP, we used CD spectroscopy performed in TFE and SDS. Additionally, previously reported NMR data, combined with ROSETTANMR, were employed to determine the structure of amidated PrRP20. The structural ensemble agrees with the spectroscopic data for the full-length peptide, which exists in an equilibrium between α- and 3(10)-helix. We demonstrate that PrRP8-20's reduced propensity to form an α-helix correlates with its reduced biological activity on mutant receptors. Further, distinct amino acid replacements in PrRP significantly decrease affinity and activity but have no influence on the secondary structure of the peptide. We conclude that formation of a primarily α-helical C-terminal region of PrRP is critical for receptor activation.
Assuntos
Hormônio Liberador de Prolactina/química , Hormônio Liberador de Prolactina/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Dicroísmo Circular , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estabilidade Proteica , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Transdução de Sinais , Dodecilsulfato de Sódio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura , Ácido Trifluoracético/químicaRESUMO
This short review aims to assess the application of basic quality assurance (QA) principles in published thyroid hormone bioanalytical methods using mass spectrometry (MS). The use of tandem MS, in particular linked to liquid chromatography has become an essential bioanalytical tool for the thyroid hormone research community. Although basic research laboratories do not usually work within the constraints of a quality management system and regulated environment, all of the reviewed publications, to a lesser or greater extent, document the application of QA principles to the MS methods described. After a brief description of the history of MS in thyroid hormone analysis, the article reviews the application of QA to published bioanalytical methods from the perspective of selectivity, accuracy, precision, recovery, instrument calibration, matrix effects, sensitivity and sample stability. During the last decade the emphasis has shifted from developing methods for the determination of L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3), present in blood serum/plasma in the 1-100 nM concentration range, to metabolites such as 3-iodo-L-thyronamine (3-T1AM), 3,5-diiodo-L-thyronine (3,5-T2) and 3,3'-diiodo-L-thyronine (3,3'-T2). These metabolites seem likely to be present in the low pM concentrations; consequently, QA parameters such as selectivity and sensitivity become more critical. The authors conclude that improvements, particularly in the areas of analyte selectivity, matrix effect measurement/documentation and analyte recovery would be beneficial.
Assuntos
Hormônios Tireóideos/análise , Tiroxina/análise , Tri-Iodotironina/análise , Animais , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Humanos , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normas , Hormônios Tireóideos/química , Tiroxina/química , Tri-Iodotironina/químicaRESUMO
Tyrosine and phenolic ring de-iodination of thyroid hormones (TH) is crucial for regulating their physiological activity. Furthermore, reactions such as de-carboxylation to thyronamines (TAM) and de-amination to thyroacetic acids (TAc) produce TH metabolites (THM) with distinct biological properties. This needs to be considered when studying effects of TH and THM. The accurate and precise quantitative analysis of TH and THM in cell culture supernatants and cell lysates are key procedures required for studying the in vitro metabolism of TH. We report here the development of a liquid-liquid extraction/isotope dilution-liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the quantification of 9 thyronines (TN) and 6 TAM in human hepatocellular carcinoma Hep G2 cell lysate extracts. In addition, we adapted the method to quantify TH, TAM and TAc, in cell lysates of FBS-depleted rat thyroid epithelium PCCL3 cells. The methods for both cell lines were validated by rigorous assessment of linearity, limits of quantification and detection (LLOQ and LLOD respectively), intra- and inter-day accuracy, precision, process efficiency (PE), matrix effect (ME) and relative recovery (RE). Calibration curves covering 11 concentrations (based on 400 µl of lysate) were linear in the range 0.016-50 nM and 0.010-50 nM for Hep G2 and PCCL3 cells respectively. The lower limits of quantification were in the range 0.031 to 1 nM. We applied the PCCL3 version of the LC-MS/MS method to the analysis of lysed cell extracts from PCCL3 cells that had been incubated with 3-iodo-L-thyronine (T1), 3-iodothyronamine (3-T1AM) and 3-iodothyroacetic acid (3-T1Ac). Over the course of 30 minutes incubation 3-T1AM was de-iodinated to 4-[4-(2-aminoethylphenoxy)]phenol (thyronamine, T0AM) and de-aminated to 3-T1Ac respectively, whilst T1 underwent de-iodination to T0. This data indicates avid metabolism of these mono-iodinated compounds and the utility of LC-MS/MS to quantify such cellular metabolism.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Iodo/metabolismo , Espectrometria de Massas/métodos , Glândula Tireoide/metabolismo , Hormônios Tireóideos/metabolismo , Linhagem Celular , Células Hep G2 , Humanos , Limite de Detecção , Controle de Qualidade , Reprodutibilidade dos Testes , Glândula Tireoide/citologiaRESUMO
Monocarboxylate transporters (MCTs) belong to the SLC16 family within the major facilitator superfamily of transmembrane transporters. MCT8 is a thyroid hormone transporter mutated in the Allan-Herndon-Dudley syndrome, a severe psychomotor retardation syndrome. MCT10 is closely related to MCT8 and is known as T-type amino acid transporter. Both transporters mediate T3 transport, but although MCT8 also transports rT3 and T4, these compounds are not efficiently transported by MCT10, which, in contrast, transports aromatic amino acids. Based on the 58% amino acid identity within the transmembrane regions among MCT8 and MCT10, we reasoned that substrate specificity may be primarily determined by a small number of amino acid differences between MCT8 and MCT10 along the substrate translocation channel. Inspecting the homology model of MCT8 and a structure-guided alignment between both proteins, we selected 8 amino acid positions and prepared chimeric MCT10 proteins with selected amino acids changed to the corresponding amino acids in MCT8. The MCT10 mutant harboring 8 amino acid substitutions was stably expressed in Madin-Darby canine kidney 1 cells and found to exhibit T4 transport activity. We then successively reduced the number of amino acid substitutions and eventually identified a minimal set of 2-3 amino acid exchanges which were sufficient to allow T4 transport. The resulting MCT10 chimeras exhibited KM values for T4 similar to MCT8 but transported T4 at a slower rate. The acquisition of T4 transport by MCT10 was associated with complete loss of the capacity to transport Phe, when Tyr184 was mutated to Phe.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Transportadores de Ácidos Monocarboxílicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Aminoácidos/química , Aminoácidos/genética , Animais , Biotinilação , Western Blotting , Linhagem Celular , Cromatografia Líquida , Cães , Transportadores de Ácidos Monocarboxílicos/genética , Mutagênese Sítio-Dirigida , Especificidade por Substrato , Espectrometria de Massas em Tandem , Hormônios Tireóideos/metabolismo , Tri-Iodotironina/metabolismo , XenopusRESUMO
A liquid-liquid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) method to determine iodothyronines and thyronamines (TAM) from cell culture media was developed. Thyroid hormones (TH) are metabolized by sequential deiodination to eventually yield thyronine (T0), but can also be decarboxylated, resulting in TAM. The method presented here for extraction of DMEM/F12 cell culture media is a fundamental procedure for a precise determination of 9 TH and 6 TAM from a single LC run. Analytes and internal standards (IS) were extracted from DMEM/F12 (w/o phenol red) by liquid-liquid extraction using isopropanol-TBME (30:70 v/v). Measurement of TH and TAM was performed during a 10-min run time using (13)C6-T4, (13)C6-T3, (13)C6-rT3, (13)C6-3,3'T2 and (2)H4-T1AM as IS. Calibration curves covered 11 calibrators measured as triplicates each for the analysis of the 9 TH and 6 TAM metabolites, and the 5 IS were linear and reproducible in the range of 0.12-120 nM (R(2) 0.991-0.999) for all calibrators. The lower limit of quantification was 0.078-0.234 nM. Method validation and robustness were demonstrated by the analysis of precision, accuracy, process efficiency, matrix effects and recoveries, as well as intra- and interassay stability. These parameters were investigated for high, middle and low concentrations of quality controls of all 9 TH and 6 TAM metabolites. This validated, sensitive and interaction-free LC-MS/MS method allows rapid analysis and accurate determination of TH and TAM from DMEM/F12 (w/o phenol red) conditioned media and seems to be easily transferable and applied to commonly used buffers and cell culture media.
RESUMO
Selenium is an essential trace element and mediates its functions via various selenoproteins such as glutathione peroxidases or thioredoxin reductases. A suboptimal selenium supply causes metabolic disturbances and is associated with an increased risk to develop different disorders, including cancer or cardiovascular diseases. This study aimed to assess the impact of a suboptimal selenium status on the hepatic metabolome of male mice analyzed by a targeted liquid chromatography/tandem mass spectrometry and a method based on non-targeted gas chromatography hyphenated with mass spectrometry. Feeding animals a diet with about half of the recommended selenium content supplied as selenomethionine caused liver glutathione peroxidase and thioredoxin reductase activities to decline and lipid peroxidation to increase. Serum T3 thyroid hormone concentration also declined via a reduced hepatic deiodinase activity. Metabolite profiling revealed predominantly changes in cysteine and carbon-1 metabolism as well as in selected lipid subclasses. In particular the concentrations of palmitoylcarnitines and oleoylcarnitines (C18:1 and C16:1) and various phosphatidylcholine species containing saturated fatty acids were elevated. Increased taurine levels suggested an enhanced cysteine flux through the salvage pathway whereas increased homocysteine levels appeared to be a consequence of a massive down-regulation of cystathionine ß lyase (cystathionine ß synthase) and a reduced flux through the transsulfuration pathway. The findings demonstrate that a suboptimal selenium status causes alterations in lipid and carbon-1 metabolism in mouse liver. These changes may contribute to the development of diseases associated with a suboptimal selenium status.
Assuntos
Fígado/metabolismo , Selênio/metabolismo , Animais , Carnitina/análogos & derivados , Carnitina/metabolismo , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , Glicogênio/metabolismo , Peroxidação de Lipídeos , Masculino , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Espectrometria de Massas em Tandem , Tri-Iodotironina/metabolismoRESUMO
The binding pocket of both NPFF receptors was investigated, focusing on subtype-selective behavior. By use of four nonpeptidic compounds and the peptide mimetics RF9 and BIBP3226, agonistic and antagonistic properties were characterized. A set of Ala receptor mutants was generated. The binding pocket was narrowed down to the upper part of transmembrane helices V, VI, VII and the extracellular loop 2. Positions 5.27 and 6.59 have been shown to have a strong impact on receptor activation and were suggested to form an acidic, negatively charged binding pocket in both NPFF receptor subtypes. Additionally, position 7.35 was identified to play an important role in functional selectivity. According to docking experiments, the aryl group of AC-216 interacts with position 7.35 in the NPFF(1) but not in the NPFF(2) receptor. These results provide distinct insights into the receptor specific binding pockets, which is necessary for the development of drugs to address the NPFF system.
Assuntos
Guanidina/química , Peptidomiméticos/farmacologia , Receptores de Neuropeptídeos/química , Adamantano/análogos & derivados , Adamantano/química , Substituição de Aminoácidos , Animais , Arginina/análogos & derivados , Arginina/química , Sítios de Ligação , Bioensaio , Células COS , Chlorocebus aethiops , Dipeptídeos/química , Desenho de Fármacos , Células HEK293 , Humanos , Ligantes , Microscopia de Fluorescência , Conformação Molecular , Peptidomiméticos/síntese química , Peptidomiméticos/química , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Selectivity is a major issue in closely related multiligand/multireceptor systems. In this study we investigated the RFamide systems of hNPFF1R and hNPFF2R that bind the endogenous peptide hormones NPFF, NPAF, NPVF, and NPSF. By use of a systematic approach, we characterized the role of the C-terminal dipeptide with respect to agonistic properties using synthesized [Xaa 7]NPFF and [Xaa 8]NPFF analogues. We were able to identify only slight differences in potency upon changing the position of Arg 7, as all modifications resulted in identical behavior at the NPFF1R and NPFF2R. However, the C-terminal Phe 8 was able to be replaced by Trp or His with only a minor loss in potency at the NPFF2R relative to the NPFF1R. Analogues with shorter side chains, such as α-amino-4-guanidino butyric acid ([Agb 7]NPFF) or phenylglycine ([Phg 8]NPFF), decreased efficacy for the NPFF1 R to 25-31 % of the maximal response, suggesting that these agonist-receptor complexes are more susceptible to structural modifications. In contrast, mutations to the conserved Asp 6.59 residue in the third extracellular loop of both receptors revealed a higher sensitivity toward the hNPFF2R receptor than toward hNPFF1R. These data provide new insight into the subtype-specific agonistic activation of the NPFF1 and NPFF(2) receptors that are necessary for the development of selective agonists.
Assuntos
Neuropeptídeos/farmacologia , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/química , Relação Estrutura-Atividade , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Humanos , Dados de Sequência Molecular , Neuropeptídeos/química , Mutação Puntual , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Alinhamento de SequênciaRESUMO
The synthesis and characterisation of the monomeric amidinato-indium(I) and thallium(I) complexes, [M(Piso)].PisoH, M = In or Tl, Piso- = [ArNC(Bu(t))NAr]-, Ar = C6H3Pr(i)2-2,6, are reported. These complexes, in which the metal centre is chelated by the amidinate ligand in an N,eta3-arene-fashion, can be considered as isomers of four-membered group 13 metal(I) carbene analogues. Theoretical studies have compared the relative energies of both isomeric forms of a model complex, [In{PhNC(H)NPh}].