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1.
Xenobiotica ; : 1-23, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38607350

RESUMO

RNA interference (RNAi) is a biological process that evolved to protect eukaryotic organisms from foreign genes delivered by viruses. This process has been adapted as a powerful tool to treat numerous diseases through the delivery of small-interfering RNAs (siRNAs) to target cells to alter aberrant gene expression.Antibody-oligonucleotide conjugates (AOCs) are monoclonal antibodies with complexed siRNA or antisense oligonucleotides (ASOs) that have emerged to address some of the challenges faced by naked or chemically conjugated siRNA, which include rapid clearance from systemic circulation and lack of selective delivery of siRNA to target cells.It is essential to characterize the ADME properties of an AOC during development to optimize distribution to target tissues, to minimize the impact of biotransformation on exposure, and to characterize the PK/PD relationship to guide translation. However, owing to the complexity of AOC structure, this presents significant bioanalytical challenges, and multiple bioanalytical measurements are required to investigate the pharmacokinetics and biotransformation of the antibody, linker, and siRNA payload.In this paper, we describe an analytical workflow that details in vivo characterization of AOCs through measurement of their distinct molecular components to provide the basis for greater understanding of their ADME properties. Although the approaches herein can be applied to in vitro characterization of AOCs, this paper will focus on in vivo applications. This workflow relies on high-resolution mass spectrometry as the principal means of detection and leverages chromatographic, affinity-based, and enzymatic sample preparation steps.

2.
Drug Metab Dispos ; 50(6): 846-857, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35306476

RESUMO

Unlike with new chemical entities, the biotransformation of therapeutic proteins (TPs) has not been routinely investigated or included in regulatory filings. Nevertheless, there is an expanding pool of evidence suggesting that a more in-depth understanding of biotransformation could better aid the discovery and development of increasingly diverse modalities. For instance, such biotransformation analysis of TPs affords important information on molecular stability, which in turn may shed light on any potential impact on binding affinity, potency, pharmacokinetics, efficacy, safety, or bioanalysis. This perspective summarizes the current practices in studying biotransformation of TPs and related findings in the biopharmaceutical industry. Various TP case studies are discussed, and a fit-for-purpose approach is recommended when investigating their biotransformation. In addition, we provide a decision tree to guide the biotransformation characterization for selected modalities. By raising the awareness of this important topic, which remains relatively underexplored in the development of TPs (Bolleddula et al., 2022), we hope that current and developing practices can pave the way for establishing a consensus on the biotransformation assessment of TPs. SIGNIFICANCE STATEMENT: This article provides a comprehensive perspective of the current practices for exploring the biotransformation of therapeutic proteins across the drug development industry. We, the participants of the Innovation and Quality therapeutic protein absorption distribution metabolism excretion working group, recommend and summarize appropriate approaches for conducting biotransformation studies to support internal decision making based on the data generated in discovery and development.


Assuntos
Produtos Biológicos , Indústria Farmacêutica , Biotransformação , Humanos
3.
Anal Chem ; 81(10): 3950-6, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19388669

RESUMO

Low-abundance protein quantification has historically been performed using ligand binding techniques. However, due to the time and cost associated with developing enzyme-linked immunosorbent assay (ELISA), mass spectrometric approaches are playing an increasingly important role. Protein quantification at or below the nanogram per milliliter level using liquid chromatography/tandem mass spectrometry (LC/MS/MS) typically utilizes an immunoaffinity (IA) enrichment step such as immunoprecipitation. In order to maximize mass spectrometry (MS) sensitivity, protein enrichment is followed by a proteolytic cleavage step used to generate a surrogate peptide with better mass spectrometric properties. Unlike ELISA, IA-LC/MS/MS is a serial technique that can require up to 3 days for a single batch analysis due to lengthy incubation and digestion steps. This report describes the use of immunoprecipitation in 96-well ELISA format (IPE) and microwave-assisted protein digestion to reduce the time required to perform LC/MS/MS protein analyses to within a single day. The utility of this approach was investigated through its application to previously published LC/MS/MS protein assays from our laboratory for two cardiotoxicity biomarkers, Myl3 and NTproBNP. Using commercially available antibodies, IPE and microwave-assisted digestion were used to repeat intraday validations for these markers, and intraday precision (%CV) and accuracy (%RE) did not exceed 11% or 3% for either assay, respectively. Additionally, lower limits of quantification of 100 pg/mL (NTproBNP) and 0.95 ng/mL (Myl3) were achieved.


Assuntos
Cromatografia Líquida/métodos , Cadeias Leves de Miosina/análise , Peptídeo Natriurético Encefálico/análise , Fragmentos de Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/imunologia , Biomarcadores/análise , Humanos , Imunoprecipitação , Micro-Ondas , Ratos
4.
Expert Rev Proteomics ; 4(2): 175-86, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17425454

RESUMO

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.


Assuntos
Proteínas/análise , Proteômica/métodos , Proteômica/normas , Anticorpos , Biomarcadores/análise , Cromatografia de Afinidade , Cromatografia Líquida , Espectrometria de Massas/métodos , Proteínas/imunologia
5.
J Chromatogr B Analyt Technol Biomed Life Sci ; 846(1-2): 359-63, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16962391

RESUMO

The ability to selectively measure serine enantiomer concentrations in rat brain microdialysate is essential during drug discovery to study the interaction of d-serine with the N-methyl-d-aspartate (NMDA) subtype of the glutamate receptor. NMDA receptor-stimulating agents, such as d-serine, have been shown to reduce the negative symptoms and cognitive dysfunction in individuals with schizophrenia when added to conventional or atypical antipsychotic drug regimens. In the work presented here, an LC/MS/MS assay was developed and validated to simultaneously measure d-serine and l-serine concentrations in rat brain microdialysate. Reverse phase chromatographic resolution of the enantiomers was obtained through derivatization with 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (Marfey's reagent). The assay was validated to determine concentrations over the range of 10-7500 ng/mL using electrospray ionization and multiple reaction monitoring (MRM). Both intra- and inter-day precision and accuracy were less than 16.5% (RE) and 7% (CV) for both analytes, respectively, and assay throughput was increased significantly relative to existing methodologies.


Assuntos
Alanina/análogos & derivados , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Dinitrobenzenos/química , Serina/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Alanina/química , Animais , Microdiálise , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estereoisomerismo
6.
MAbs ; 9(2): 285-296, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27981884

RESUMO

A recent report described a novel mechanism of action for an anti-proprotein convertase subtilisin-kexin type 9 (PCSK9) monoclonal antibody (LY3015014, or LY), wherein the antibody has improved potency and duration of action due to the PCSK9 epitope for LY binding. Unlike other antibodies, proteolysis of PCSK9 can occur when LY is bound to PCSK9. We hypothesized that this allowance of PCSK9 cleavage potentially improves LY efficiency through two pathways, namely lack of accumulation of intact PCSK9 and reduced clearance of LY. A quantitative modeling approach is necessary to further understand this novel mechanism of action. We developed a mechanism-based model to characterize the relationship between antibody pharmacokinetics, PCSK9 and LDL cholesterol levels in animals, and used the model to better understand the underlying drivers for the improved efficiency of LY. Simulations suggested that the allowance of cleavage of PCSK9 resulting in a lack of accumulation of intact PCSK9 is the major driver of the improved potency and durability of LY. The modeling reveals that this novel 'proteolysis-permitting' mechanism of LY is a means by which an efficient antibody can be developed with a total antibody dosing rate that is lower than the target production rate. We expect this engineering approach may be applicable to other targets and that the mathematical models presented herein will be useful in evaluating similar approaches.


Assuntos
Anticorpos Monoclonais/farmacocinética , Modelos Teóricos , Inibidores de PCSK9 , Animais , LDL-Colesterol/metabolismo , Humanos , Macaca fascicularis , Camundongos , Proteólise/efeitos dos fármacos
7.
Bioanalysis ; 8(15): 1579-1595, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27314304

RESUMO

BACKGROUND: A thorough understanding of the biological role of oxyntomodulin (OXM) has been limited by the availability of sensitive and specific analytical tools for reliable in vivo characterization. Here, we utilized immunoaffinity capture coupled with high-resolution accurate mass LC-MS detection to quantify OXM and its primary catabolites. RESULTS: Quantification of intact OXM 1-37 in human and rat plasma occurred in pre- and post-prandial samples. Profiles for the major catabolites were observed allowing kinetic differences to be assessed between species. CONCLUSION: A validated assay in human and rat plasma was obtained for OXM 1-37 and its catabolites, 3-37 and 4-37. The value of full scan high-resolution accurate mass detection without selected reaction monitoring for low-abundance peptide quantification was also demonstrated.


Assuntos
Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Oxintomodulina/sangue , Animais , Humanos , Limite de Detecção , Masculino , Ratos , Ratos Sprague-Dawley
8.
Curr Top Med Chem ; 2(1): 53-66, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11899065

RESUMO

LC/MS/MS based bioanalysis using atmospheric pressure ionization (API)-style interfaces has now been applied for over a decade. This technology, which initially found application for clinical bioanalysis, is now firmly established as the primary bioanalytical tool for ADME studies related to drug discovery and lead optimization (LO). This review focuses on recent advances in LC/MS/MS based bioanalysis in support of drug discovery and LO. The initial part of the article reviews the principal components of LC/MS/MS bioanalysis: sample preparation, chromatography, ionization and mass analysis. In each section, factors affecting high throughput bioanalysis are addressed. Because of the importance of on-line column switching methods to discovery bioanalysis, the section on sample preparation is divided into off-line and on-line approaches. In addition, the discussion of chromatography is limited to reversed phase liquid chromatography with emphasis given to the trend towards high-flow gradient elution techniques. The latter part of the review focuses on considerations for experimental design. In this section, pooling methods such as cassette dosing are discussed along with more highly integrated strategies linking bioanalysis with protocol generation and sample collection. The article concludes by briefly reviewing factors, which affect bioanalytical precision and accuracy, such as ion suppression, analyte stability and metabolite interference.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Espectrometria de Massas/métodos , Animais , Cromatografia Líquida de Alta Pressão , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Humanos , Espectrometria de Massas/instrumentação , Manejo de Espécimes , Tecnologia Farmacêutica/instrumentação , Tecnologia Farmacêutica/métodos
9.
Mol Biosyst ; 10(7): 1730-41, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24671508

RESUMO

The phosphatase Rtr1 has been implicated in dephosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) during transcription elongation and in regulation of nuclear import of RNAPII. Although it has been shown that Rtr1 interacts with RNAPII in yeast and humans, the specific mechanisms that underlie Rtr1 recruitment to RNAPII have not been elucidated. To address this, we have performed an in-depth proteomic analysis of Rtr1 interacting proteins in yeast. Our studies revealed that hyperphosphorylated RNAPII is the primary interacting partner for Rtr1. To extend these findings, we performed quantitative proteomic analyses of Rtr1 interactions in yeast strains deleted for CTK1, the gene encoding the catalytic subunit of the CTD kinase I (CTDK-I) complex. Interestingly, we found that the interaction between Rtr1 and RNAPII is decreased in ctk1Δ strains. We hypothesize that serine-2 CTD phosphorylation is required for Rtr1 recruitment to RNAPII during transcription elongation.


Assuntos
Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Fatores de Transcrição/metabolismo , Domínio Catalítico , Fosforilação , Proteômica , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
10.
Toxicol Sci ; 114(2): 183-92, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20044590

RESUMO

Chronic treatment with suprapharmacologic doses of peroxisome proliferator-activated receptor (PPAR) agonists has a known potential for causing left ventricular hypertrophy (LVH). The mechanism by which LVH develops is not well understood nor are biomarkers of it well characterized. Natriuretic peptides are important regulators of cardiac growth, blood volume, and arterial pressure and may be useful biomarkers of LVH and hemodynamic changes that precede it. We measured amino-terminal pro-atrial natriuretic peptide (NTproANP), amino-terminal pro-brain natriuretic peptide (NTproBNP), and cardiac troponin I (cTnI) concentrations in serum and plasma, as well as transcripts in left ventricular heart tissue for atrial natriuretic peptide precursor (Nppa), brain natriuretic peptide precursor (Nppb), and myosin heavy chain-beta (Myh7) as potential biomarkers of LVH induced by a PPARalpha/gamma dual agonist in Sprague-Dawley rats. We used magnetic resonance imaging, echocardiography, and hemodynamics to identify structural and functional cardiovascular changes related to the biomarkers. Heart-to-brain weight ratios (HW:BrW) were correlated with NTproANP, NTproBNP, and cTnI concentrations in serum as well as fold change in expression of Nppa and Nppb. LVH was characterized by increased left ventricular wall thickness and inner diameter, increased cardiac output, decreased arterial blood pressure, and increased heart rate. In these studies, each end point contributed to the early detection of LVH, the ability to monitor its progression, and demonstrated the ability of NTproANP concentration in serum to predict LVH and hemodynamic changes.


Assuntos
Fármacos Cardiovasculares/toxicidade , Hipertrofia Ventricular Esquerda/diagnóstico , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/toxicidade , Tiofenos/toxicidade , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Biomarcadores/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Feminino , Coração/efeitos dos fármacos , Hipertrofia Ventricular Esquerda/induzido quimicamente , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Imageamento por Ressonância Magnética , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Peptídeo Natriurético Encefálico/genética , Peptídeo Natriurético Encefálico/metabolismo , Tamanho do Órgão/efeitos dos fármacos , PPAR alfa/metabolismo , PPAR gama/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Troponina T/genética , Troponina T/metabolismo
11.
Anal Chem ; 80(3): 561-6, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18179251

RESUMO

Brain natriuretic peptide (BNP) and N-terminal proBNP (NTproBNP) are well established in the clinic as biomarkers of heart failure. BNP hormone and the inactive NTproBNP are predominantly secreted in the ventricles of the heart in response to pressure overload and, consequently, are being investigated as markers of drug-induced cardiac hypertrophy in rat to support drug development. In the work presented here, an immunoaffinity-based LC/MS/MS assay was developed and validated to measure a selective tryptic fragment of NTproBNP in rat serum. The assay covers the range of 13-329 pg/mL of the tryptic fragment LLELIR, corresponding to 0.1-2.5 ng/mL intact NTproBNP. A stable isotope-labeled version of NTproBNP containing the tryptic fragment LLELI[13C615N1]R was prepared by solid-phase peptide synthesis and was used as an internal standard to minimize assay variability. Due to endogenous NTproBNP present in rat serum, human serum was used as the control matrix, and parallelism between rat and human serum was established by standard addition. Assay accuracy (% RE) and precision (% CV) were measured at three concentrations on each of 4 days and did not exceed 4.2 and 14.5%, respectively. Additionally, study data are presented from the application of this assay in which rats demonstrated a significant increase in NTproBNP serum concentration following administration of an agent known to induce cardiac hypertrophy. In this study, the relationship between serum NTproBNP and cardiac hypertrophy was corroborated by increases in heart weight and magnetic resonance imaging of the test subjects' left ventricle. To our knowledge, this represents the first reported assay for NTproBNP in preclinical species for the assessment of cardiac hypertrophy.


Assuntos
Cardiomiopatia Hipertrófica/induzido quimicamente , Cromatografia Líquida/métodos , Imunoprecipitação/métodos , Espectrometria de Massas/métodos , Peptídeo Natriurético Encefálico/sangue , Sequência de Aminoácidos , Animais , Biomarcadores/sangue , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Modelos Animais de Doenças , Humanos , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
12.
Artigo em Inglês | MEDLINE | ID: mdl-20636083

RESUMO

Current drug discovery involves a highly iterative process pertaining to three core disciplines: biology, chemistry, and drug disposition. For most pharmaceutical companies the path to a drug candidate comprises similar stages: target identification, biological screening, lead generation, lead optimization, and candidate selection. Over the past decade, the overall efficiency of drug discovery has been greatly improved by a single instrumental technique, liquid chromatography/mass spectrometry (LC/MS). Transformed by the commercial introduction of the atmospheric pressure ionization interface in the mid-1990s, LC/MS has expanded into almost every area of drug discovery. In many cases, drug discovery workflow has been changed owing to vastly improved efficiency. This review examines recent trends for these three core disciplines and presents seminal examples where LC/MS has altered the current approach to drug discovery.


Assuntos
Química Farmacêutica/tendências , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Preparações Farmacêuticas/análise , Animais , Técnicas de Química Analítica , Formas de Dosagem , Desenho de Fármacos , Descoberta de Drogas , Indústria Farmacêutica/tendências , Humanos
13.
Anal Chem ; 79(11): 4199-205, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17447729

RESUMO

Myosin light chain 1 (Myl3) is a 23-kDa isoform of one of the subunits of myosin, a protein involved in muscle contraction. Myl3 is presently being studied as a biomarker of cardiac necrosis to predict drug-induced cardiotoxicity, and in the work presented here, an LC/MS/MS assay was developed and validated to measure Myl3 in rat serum. The key steps in this approach involved immunoaffinity purification of Myl3 from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified using a synthetic peptide standard and a corresponding stable isotope-labeled internal standard, and the results were stoichiometrically converted to Myl3 serum concentrations. Myl3 concentrations were corrected for peptide recovery following immunoprecipitation and digestion (85%) and showed excellent agreement with synthetic peptide standards. Both the synthetic peptide and His-Myl3 protein were used to evaluate assay accuracy (% RE) and precision (% CV), which were measured on each of 3 days. The synthetic peptide was evaluated over the range of 0.073-7.16 nM, while Myl3 protein QC samples prepared in rat serum were evaluated over the range of 0.13-6.62 nM. To prepare control matrix, endogenous Myl3 was immunodepleted from pooled rat serum. Peptide interday accuracy and precision did not exceed 7.6 and 11.1%, and Myl3 interday accuracy and precision did not exceed 12.9 and 13.2%, respectively. Data are presented from the application of this assay to establish a time course in which rats demonstrated a marked increase in Myl3 serum concentrations following administration of isoproterenol, a beta-adrenergic receptor agonist known to induce cardiac injury. This assay is an example of a larger effort in our laboratory to use LC/MS/MS in conjunction with immunoaffinity techniques to evaluate candidate biomarkers of target organ toxicity and to expedite the development of biomarker assays for drug development.


Assuntos
Cromatografia Líquida/métodos , Cardiopatias/metabolismo , Imunoprecipitação/métodos , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Biomarcadores/química , Biomarcadores/metabolismo , Isoproterenol , Dados de Sequência Molecular , Necrose/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley
14.
Proteomics Clin Appl ; 1(7): 661-71, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21136721

RESUMO

Heart fatty acid binding protein (Fabp3) is a cytosolic protein expressed primarily in heart, and to a lesser extent in skeletal muscle, brain, and kidney. During myocardial injury, the Fabp3 level in serum is elevated rapidly, making it an ideal early marker for myocardial infarction. In this study, an MS-based selected reaction monitoring method (LC-SRM) was developed for quantifying Fabp3 in rat serum. Fabp3 was enriched first through an immobilized antibody, and the protein was digested on beads directly. A marker peptide of Fabp3 was quantified using LC-SRM with a stable isotope-labeled peptide standard. For six quality control samples with Fabp3 ranging from 0.256 to 25 ng, the average recovery following the procedure was about 73%, and the precision (%CV) between replicates was less than 7%. The Fabp3 concentrations in rat serum peaked 1 h after isoproterenol treatment, and returned to baseline levels 24 h after the dose. Elevated Fabp3 levels were also detected in rats administered with a PPAR α/δ agonist, which has shown to cause skeletal muscle necrosis. Fabp3 can be used as a biomarker for both cardiac and skeletal necroses. The cross-validation of the LC-SRM method with an existing ELISA method is described.

15.
Anal Biochem ; 356(2): 235-43, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16797470

RESUMO

Proteolytic fragments of type II collagen, a major component of joint tissue, have recently been identified as biomarkers for osteoarthritis, a progressive disease associated with cartilage degeneration. A liquid chromatography/tandem mass spectrometry (MS/MS) assay that utilizes online immunoaffinity chromatography and column switching was developed in our laboratory for the neoepitope of type II collagen (NET2C). During method development, peptide collision-induced dissociation (CID) was found to be a significant source of assay variation, which exceeded 10% CV, despite the fact that a stable-isotope-labeled (SIL) internal standard was used to minimize imprecision. This phenomenon was studied in detail using peptides and associated SIL internal standards of varying lengths and amino acid compositions. Variability in peptide CID necessitated the monitoring of multiple MS/MS transitions to obtain acceptable assay precision. The assay was subsequently validated to measure NET2C concentrations in rat urine over the range of 0.1 to 10 ng/mL. The interday accuracy and precision ranged from 3.9 to 13.1 (%CV) and 10.7 to 5.3 (%RE), respectively, across the range of validated concentrations. A specific application of the assay is presented in which the role of estrogen deficiency in the development and progression of osteoarthritis was investigated. In this study, the effect of estrogen on lowering NET2C concentrations in urine in ovariectomized rats was demonstrated.


Assuntos
Biomarcadores/urina , Cromatografia Líquida de Alta Pressão/métodos , Colágeno Tipo II/urina , Espectrometria de Massas/métodos , Peptídeos/urina , Animais , Biomarcadores/química , Colágeno Tipo II/química , Feminino , Peptídeos/química , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes
16.
Rapid Commun Mass Spectrom ; 20(24): 3723-35, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17117458

RESUMO

The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Cromatografia Líquida/métodos , Imunoensaio/métodos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Espectrometria de Massas por Ionização por Electrospray/métodos , Biomarcadores/líquido cefalorraquidiano , Humanos , Técnica de Diluição de Radioisótopos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Rapid Commun Mass Spectrom ; 16(6): 537-43, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-11870891

RESUMO

The effects of flow rate and column length on analyte response (peak area and height), total cycle time, column backpressure, and elution volume are presented. Rapid chromatographic separations and tandem mass spectrometric (MS/MS) detection are applied to the supernatant of protein-precipitated plasma standards containing four compounds from a drug discovery screen. The plasma samples were injected onto three C-18 columns (2 x 10,2.1 x 30 and 2.1 x 50 mm) at flow rates of 0.25, 0.50, 1.00 and 1.50 mL/min. The plasma samples were detected using a Sciex API 3000 tandem mass spectrometer operated in the Turbo Ionspray mode. A post-column split was used to maintain a flow rate of 0.25 mL/min into the mass spectrometer source to avoid differences in nebulization efficiency. The data show that diluted protein-precipitated plasma supernatants show average matrix effects (i.e. suppression) of 60.0% (2 x 10 mm), 89.3% (2 x 30 mm), and 76.7% (2 x 50 mm) of expected response at 10 ng/mL. Average matrix effects of 70.2% (2 x 10 mm), 88.9% (2 x 30 mm), and 81.2% (2 x 50 mm) of expected response at 1000 ng/mL plasma. The data also show if peak widths remain relatively constant, analytes are less sensitive as flow rates are increased. These data are consistent with the concentration-dependent relationship of ionspray in the range of flow rates studied. The data show that, while analyte response decreased proportionately to increases in flow rate, the analysis cycle times did not decrease proportionately.


Assuntos
Proteínas Sanguíneas/análise , Cromatografia Líquida de Alta Pressão/instrumentação , Espectrometria de Massas/instrumentação , Precipitação Química , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Espectrometria de Massas/métodos
18.
J Pharmacol Exp Ther ; 301(3): 1020-4, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12023533

RESUMO

The first endocannabinoid, anandamide, was discovered in 1992. Since then, two other endocannabinoid agonists have been identified, 2-arachidonyl glycerol and, more recently, noladin ether. Here, we report the identification and pharmacological characterization of a novel endocannabinoid, virodhamine, with antagonist properties at the CB1 cannabinoid receptor. Virodhamine is arachidonic acid and ethanolamine joined by an ester linkage. Concentrations of virodhamine measured by liquid chromatography atmospheric pressure chemical ionization-tandem mass spectrometry in rat brain and human hippocampus were similar to anandamide. In peripheral tissues that express the CB2 cannabinoid receptor, virodhamine concentrations were 2- to 9-fold higher than anandamide. In contrast to previously described endocannabinoids, virodhamine was a partial agonist with in vivo antagonist activity at the CB1 receptor. However, at the CB2 receptor, virodhamine acted as a full agonist. Transport of [(14)C]anandamide by RBL-2H3 cells was inhibited by virodhamine. Virodhamine produced hypothermia in the mouse and acted as an antagonist in the presence of anandamide both in vivo and in vitro. As a potential endogenous antagonist at the CB1 receptor, virodhamine adds a new form of regulation to the endocannabinoid system.


Assuntos
Ácidos Araquidônicos/química , Canabinoides/química , Receptor CB2 de Canabinoide , Receptores de Droga/antagonistas & inibidores , Receptores de Droga/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Moduladores de Receptores de Canabinoides , Canabinoides/metabolismo , Canabinoides/farmacologia , Relação Dose-Resposta a Droga , Endocanabinoides , Etanolamina/química , Etanolamina/metabolismo , Etanolamina/farmacologia , Hipocampo/química , Hipocampo/metabolismo , Masculino , Camundongos , Alcamidas Poli-Insaturadas , Ratos , Ratos Wistar , Receptores de Canabinoides
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