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1.
Bioanalysis ; 13(3): 147-159, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33543654

RESUMO

Background: Surrogate monoclonal antibodies (mAbs) used in preclinical in vivo studies can be challenging to quantify due to lack of suitable immunoaffinity reagents or unavailability of the mAb protein sequence. Generic immunoaffinity reagents were evaluated to develop sensitive LC-MS/MS assays. Peptides of unknown sequence can be used for selective LC-MS quantification. Results: anti-mouse IgG1 was found to be an effective immunoaffinity reagent, enabling quantification of mouse IgG1 mAbs in mouse serum. Selective peptides of unknown sequence were applied for multiplex LC-MS quantification of two rat mAbs co-dosed in mouse. Conclusion: Generic anti-mouse IgG subtype-specific antibodies can be used to improve assay sensitivity and peptides of unknown sequence can be used to quantify surrogate mAbs when the mAb protein sequence in unavailable.

2.
Rapid Commun Mass Spectrom ; 35(7): e9041, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33415785

RESUMO

RATIONALE: Gangliosides (GS) are attractive targets in biomarker discovery because of their physiological significance in numerous human diseases including certain cancers and developmental and metabolic disorders. The robust strategy described here enables the profiling of numerous GS while obtaining quantitative data of exploratory biomarkers present in human plasma and whole blood. METHOD: The GS from human blood, human plasma, and several cell lines were extracted using a mixture of methanol and isopropanol/0.1% formic acid followed by direct analysis of the supernatant. The simultaneous Qualitative and Quantitative (Qual/Quan) approach involves micro flow (20 µL/min) high pressure liquid chromatography (HPLC)/high-resolution mass spectrometry (HRMS) and post-acquisition data processing with Skyline software for profiling numerous GS in biological matrices. The quantitative assay involves reverse-phase liquid chromatography/HRMS and calibration curves using commercially available GS. RESULTS: Protein precipitation resulted in ~60%-80% GS recovery from biological matrices. Direct injection of the extract allowed for quantification of targeted GS in human blood, plasma, and cancer cell lines. The lower limit of detection for the target analytes, GM1, GT1, GD1, spiked into 1% BSA/PBS, ranged from 1 to 10 ng/mL. Human lung cancer cell lines contained variable amounts (1-130 ng/mL) of soluble Fuc-GM1 analogs, potential biomarkers of lung cancer. CONCLUSIONS: A combination of simple extraction and micro-HPLC/HRMS allowed for quantification of GS in human serum and whole blood. Integration of HRMS with Skyline allowed for GS profiling in the same samples using post-acquisition HRMS data without the need for reanalysis. The strategy presented here is expected to play an important role in profiling exploratory GS biomarkers in discovery bioanalytical research.

3.
Bioanalysis ; 12(20): 1439-1447, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33006478

RESUMO

Aim: There is little information in the literature regarding assays for measuring CDH17 in tissues. Numerous studies indicate overexpression of CDH17 in a variety of diseases including hepatocellular carcinoma, colorectal and gastric cancer. Here we present an immunoaffinity enrichment LC-MS/MS approach for analysis of CDH17 in human tissues, plasma and serum as well as preclinical models. Results: CDH17 levels were measured in colon and ileum tissues from healthy donors and inflamed tissues from patients with Ulcerative Colitus or Crohn's disease. Applicability of the immunocapture LC-MS/MS approach is demonstrated for colon tissues from non-diseased mouse and cynomolgus monkey. Conclusion: The analytical approaches discussed here are suitable for quantitation of CDH17 in various tissues to enable both preclinical and clinical assessment.

4.
Anal Chem ; 92(19): 13525-13531, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32926623

RESUMO

Bioanalysis of polar analytes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains a significant challenge because of their poor chromatographic retention on the commonly used reversed-phase LC columns and the resulting severe ionization suppression from coeluting matrix components. Here we present a novel approach to perform ultrahigh-throughput and chromatography-free bioanalysis of polar compounds using a prototype acoustic ejection mass spectrometer (AEMS) platform. Previously developed for direct analysis of solid or liquid samples by MS, the open port interface (OPI) has recently been modified and coupled to an acoustic nanoliter dispenser to enable high-speed direct MS analysis from 384-well plates with a reported speed as fast as 0.5 s/sample. Ionization suppression was reduced due to the >1000 fold dilution of the original sample by the carrier solvent in the AE-OPI-MS operation. Taking full advantage of the chromatography-free and suppression-reducing features of this prototype instrument, we successfully demonstrated the ultrahigh-throughput bioanalysis of metformin, a small polar substrate commonly used in high-throughput in vitro transporter inhibition assays in the early ADME profiling space in drug discovery. The AEMS platform achieved a speed of 2.2 s/sample using only 10 nL of sample volume. Similar bioanalytical and biological results from actual assay samples were obtained by AEMS when compared to those obtained by the fastest LC-MS/MS method previously reported, along with a 15-fold speed advantage and ∼500-fold less sample consumption to enable future assay miniaturization. The general applicability of this novel approach to bioanalysis of several classes of polar analytes including ethambutol, isoniazid, ephedrine, and gemcitabine in biological matrices was further demonstrated.

5.
Bioanalysis ; 12(18): 1311-1324, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32945691

RESUMO

Background: S1PR1, a G protein-coupled receptor (GPCR) protein, is a therapeutic target for treatment of autoimmune diseases. As a potential biomarker for drug effect and patient stratification, it is of great significance to measure it in biological samples. However, due to the hydrophobic nature of S1PR1 and the difficulties in extraction and solubilization, as well as low expression levels, quantitative determination of S1PR1 remains challenging. Results: In this work, a peptide immunoaffinity LC-MS/MS method was developed to quantify S1PR1 in biopsy-sized colon samples with an LLOQ of 7.81 pM. Conclusion: Peptide immunoaffinity LC-MS/MS based strategy has achieved the desired sensitivity for low abundance S1PR1, and the same strategy could be applied to quantify S1PR1 in multiple species and other GPCR proteins.

6.
Anal Biochem ; 602: 113766, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32389692

RESUMO

The S100A1 protein is a target of interest for the treatment of heart failure as it has been previously reported to be depleted in failing cardiomyocytes. A gene therapy approach leading to increased expression levels of the protein directly in the heart could potentially lead to restoration of contractile function and improve overall cell survival. S100A1 is a relatively small soluble protein that is extremely well conserved across species with only a single amino acid difference between the sequences in human and pig, a commonly used pre-clinical model for evaluation of efficacy, biodistribution and safety for cardiac-directed gene therapy approaches. This high homology presents a bioanalytical challenge for the accurate detection and quantitation of both endogenous (pig) and exogenous (human) transduced S100A1 proteins post treatment using a human S100A1 gene therapy in pigs. Here we present a sensitive and selective LC-MS/MS approach that can easily differentiate and simultaneously quantitate both human and pig S100A1 proteins. Additionally, we report on a detailed profiling of S100A1 protein in various pig tissues, a comprehensive evaluation of S100A1 distribution in pig hearts and a comparison to S100A1 levels in human cardiac samples.

7.
Clin Chem ; 66(2): 282-301, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32040572

RESUMO

Immunoaffinity-mass spectrometry (IA-MS) is an emerging analytical genre with several advantages for profiling and determination of protein biomarkers. Because IA-MS combines affinity capture, analogous to ligand binding assays (LBAs), with mass spectrometry (MS) detection, this platform is often described using the term hybrid methods. The purpose of this report is to provide an overview of the principles of IA-MS and to demonstrate, through application, the unique power and potential of this technology. By combining target immunoaffinity enrichment with the use of stable isotope-labeled internal standards and MS detection, IA-MS achieves high sensitivity while providing unparalleled specificity for the quantification of protein biomarkers in fluids and tissues. In recent years, significant uptake of IA-MS has occurred in the pharmaceutical industry, particularly in the early stages of clinical development, enabling biomarker measurement previously considered unattainable. By comparison, IA-MS adoption by CLIA laboratories has occurred more slowly. Current barriers to IA-MS use and opportunities for expanded adoption are discussed. The path forward involves identifying applications for which IA-MS is the best option compared with LBA or MS technologies alone. IA-MS will continue to benefit from advances in reagent generation, more sensitive and higher throughput MS technologies, and continued growth in use by the broader analytical community. Collectively, the pursuit of these opportunities will secure expanded long-term use of IA-MS for clinical applications.


Assuntos
Cromatografia Líquida/métodos , Imunoensaio/métodos , Espectrometria de Massas em Tandem/métodos , Bioensaio , Biomarcadores/análise , Humanos , Proteínas/análise , Sensibilidade e Especificidade
9.
Bioanalysis ; 9(20): 1573-1588, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29072496

RESUMO

AIM: IP-10 is a protein target for the treatment of Crohn's disease. Inhibition of IP-10 by anti-IP-10 mAbs neutralizes its various biological activities. The measurement of free IP-10 suppression as a target engagement biomarker is required for the assessment of drug effect on the target. RESULTS: The development of highly sensitive immunoaffinity-LC-MS/MS assays for quantifying free and total IP-10 in cynomolgus monkey serum is reported for the first time. This paper details strategies for maximizing assay sensitivity by selecting digestion routes, and optimizing immunocapture to achieve full recovery and minimal matrix effect. For the free IP-10 assay, bioanalytical strategies have been established to minimize drug/ligand dissociation. CONCLUSION: The assays have been implemented for target engagement measurement, pharmacokinetic-pharmacodynamic correlation, and human dose projections.


Assuntos
Biomarcadores/sangue , Quimiocina CXCL10/sangue , Cromatografia de Afinidade , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Cromatografia Líquida de Alta Pressão , Humanos , Ligantes , Macaca fascicularis , Peptídeos/química , Peptídeos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
10.
Bioanalysis ; 8(15): 1611-1622, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27397670

RESUMO

BACKGROUND: Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes. RESULTS: To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC-MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences. CONCLUSION: IP-LC-MS can be applied to simultaneously quantify in vivo drug concentrations and measure the extent of isomerization or deamidation in PK studies conducted during the drug discovery stage.


Assuntos
Anticorpos Monoclonais/química , Asparagina/análise , Ácido Aspártico/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Cromatografia Líquida/métodos , Humanos , Imunoprecipitação/métodos , Isomerismo , Macaca fascicularis , Masculino , Espectrometria de Massas em Tandem/métodos
11.
Bioanalysis ; 8(13): 1383-401, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27277879

RESUMO

BACKGROUND: Antibody-drug conjugates (ADCs) are complex drug constructs with multiple species in the heterogeneous mixture that contribute to their efficacy and toxicity. The bioanalysis of ADCs involves multiple assays and analytical platforms. METHODS: A series of ligand binding and LC-MS/MS (LB-LC-MS/MS) hybrid assays, through different combinations of anti-idiotype (anti-Id), anti-payload, or generic capture reagents, and cathepsin-B or trypsin enzyme digestion, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays, total-antibody and conjugated-antibody. RESULTS & CONCLUSION: Hybrid assays are complementary or viable alternatives to ligand-binding assay for ADC bioanalysis and PK/PD modeling. The fit-for-purpose choice of analytes, assays and platforms and an integrated strategy from Discovery to Development for ADC PK and bioanalysis are recommended.


Assuntos
Imunoconjugados/sangue , Preparações Farmacêuticas/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia Líquida/métodos , Haplorrinos , Humanos , Imunoensaio/métodos , Imunoconjugados/análise , Limite de Detecção , Preparações Farmacêuticas/análise , Ratos
12.
Anal Biochem ; 503: 71-8, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27033006

RESUMO

The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC-MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC-MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time.


Assuntos
Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/metabolismo , Marcação por Isótopo , Metabolômica , Animais , Ácidos e Sais Biliares/química , Cromatografia Líquida , Humanos , Espectrometria de Massas , Estrutura Molecular , Ratos
13.
Bioanalysis ; 8(4): 265-74, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26807991

RESUMO

BACKGROUND: A target protein-based affinity extraction LC-MS/MS method was developed to enable plasma level determination following ultralow dosing (0.1-3 µg/kg) of an inhibitor of apoptosis proteins molecule. Methodology & results: Affinity extraction (AE) utilizing immobilized target protein BIR2/BIR3 was used to selectively capture the inhibitor of apoptosis proteins molecule from dog plasma and enable removal of background matrix components. Pretreatment of plasma samples using protein precipitation was found to provide an additional sensitivity gain. A LLOQ of 7.8 pM was achieved by combining protein precipitation with AE. The method was used to support an ultralow dose dog toxicity study. CONCLUSION: AE-LC-MS/MS, utilizing target protein, is a highly sensitive methodology for small molecule quantification with potential for broader applicability.


Assuntos
Análise Química do Sangue/métodos , Fracionamento Químico/métodos , Cromatografia Líquida/métodos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Isoquinolinas/análise , Limite de Detecção , Oligopeptídeos/análise , Bibliotecas de Moléculas Pequenas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Cães , Feminino , Humanos , Proteínas Imobilizadas/antagonistas & inibidores , Proteínas Imobilizadas/química , Proteínas Inibidoras de Apoptose/química , Isoquinolinas/química , Isoquinolinas/farmacologia , Masculino , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
14.
Bioanalysis ; 8(3): 193-204, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26811930

RESUMO

BACKGROUND: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well. RESULTS: For the quantitation of BMS-986089, both accuracy (99-103%) and precision (2.4-12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100-108%) and precision (0.7-18%) that were obtained for the analysis of the surrogate peptide (VVSVLTVLHQDWLNGK). For Myostatin, accuracy (94-103%) and precision (2.4-14.9%) were obtained for the analysis of the surrogate peptide (IPAMVVDR). CONCLUSION: The developed method was applied to the analysis of samples following dosing of BMS-986089 to mice. This method highlights the potential of LC-MS/MS-based methods to eventually assess in vivo drug-target engagement.


Assuntos
Cromatografia Líquida/métodos , Miostatina/análise , Proteínas Recombinantes de Fusão/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida/economia , Análise Custo-Benefício , Humanos , Imunoglobulina G/análise , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miostatina/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Ratos , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Espectrometria de Massas em Tandem/economia , Tripsina/metabolismo
15.
Bioanalysis ; 7(19): 2501-13, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26466806

RESUMO

The analysis of endogenous and exogenous analytes in biological matrices presents several challenges to the bioanalyst. These analytes are often present at low concentrations, typically in complex matrices, and may have physicochemical properties that are not amenable to LC-MS analysis. The bioanalyst thus relies heavily on the formation of analyte derivatives for the efficient quantification of these compounds. These derivatives are also critically employed to derive information on the biology of living systems, potential drug or disease targets, and biomarkers of drug efficacy, safety, or disease progression. In this perspective, we demonstrate how analyte derivatives are applied in modern bioanalytical workflows and we discuss the potential use of these derivatives in the future.


Assuntos
Biomarcadores/análise , Preparações Farmacêuticas/análise , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Humanos , Marcação por Isótopo , Preparações Farmacêuticas/química
16.
Bioanalysis ; 6(13): 1795-811, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25157486

RESUMO

BACKGROUND: The disease state can modulate the penetration of large antibody-sized therapeutic molecules into affected tissues. Suitable bioanalytical methods are required for the quantitative analysis of drug tissue levels to enable a better understanding of the parameters influencing drug penetration and target engagement. RESULTS: Described is a sensitive and selective LC-MS/MS assay for the quantification of human mAb molecules in mouse tissues. By homogenizing tissues directly into serum, a common serum calibration curve can be used for multiple tissues. A generic procedure was used for affinity enrichment. An analytical range of 20 - 20,000 ng/ml was achieved in serum. CONCLUSION: The method described here can be applied for the quantitative analysis of mAb and Fc-fusion therapeutic molecules in a variety of animal tissue matrices.


Assuntos
Anticorpos Monoclonais/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/análise , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/metabolismo , Cromatografia de Afinidade , Feminino , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/análise , Análise de Regressão , Pele/metabolismo , Tripsina/metabolismo , Ustekinumab
17.
Bioanalysis ; 6(13): 1827-41, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25157488

RESUMO

Both LBAs and LC-MS-based assays are reviewed and summarized for applications in quantitative protein analysis. A strategy for platform selection is proposed based on several factors that contribute to the complexities of bioanalysis of biologics. Protein types, multiple co-existing forms, post-translational modifications, and affinities to ADA, targets, and endogenous proteins need to be considered when selecting the most appropriate platform. Other factors, such as intended use of data, assay sensitivity, available reagents, and multiple analytes also impact on the choice of bioanalytical platform. At BMS, strategies for the seamless integration of both platforms are being implemented to provide not only PK/PD data of the molecules but also useful information of the amino acid structure and functional relationship of the proteins.


Assuntos
Cromatografia Líquida de Alta Pressão , Ligantes , Proteínas/análise , Espectrometria de Massas em Tandem , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Imunoprecipitação , Leptina/análise , Leptina/farmacocinética , Proteínas/isolamento & purificação , Proteínas/farmacocinética
18.
Bioanalysis ; 6(14): 1901-5, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25158962

RESUMO

BACKGROUND: (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid, also known as LY379268, a group II metabotropic glutamate receptor agonist, has been widely used in neuroscience as a model compound in studies evaluating antipsychotic drugs for the treatment of schizophrenia. MATERIALS & METHODS: So far, no reports describing methods of the bioanalysis of LY379268 have been published. Here, a novel method is presented for determining LY379268 in rat plasma employing precolumn derivatization with pentafluorobenzoyl chloride reagent coupled to liquid chromatography/mass spectrometry. CONCLUSION: Chemical derivatization of a low-molecular-weight and highly polar molecule yields a derivative that is retained on a reversed-phase liquid chromatography column with improved tandem mass spectrometric response.


Assuntos
Aminoácidos/sangue , Compostos Bicíclicos Heterocíclicos com Pontes/sangue , Cromatografia Líquida/métodos , Receptores de Glutamato Metabotrópico/agonistas , Espectrometria de Massas em Tandem/métodos , Aminoácidos/química , Animais , Benzoatos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Cromatografia de Fase Reversa/métodos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Ratos
19.
Rapid Commun Mass Spectrom ; 28(13): 1535-43, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24861605

RESUMO

RATIONALE: Liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays are increasingly being used for absolute quantitation of proteins due to high specificity and low cost. However, the major challenge for the LC/MS method is insufficient sensitivity. This paper details the strategies developed to maximize the sensitivity from aspects of chromatography, mass spectrometry, and sample preparation to achieve a highly sensitive LC/MS method. METHODS: The method is based on the LC/MS/MS measurement of a surrogate peptide generated from trypsin digestion of interferon-gamma-inducible protein-10 (IP-10). The sample preparation strategy involved selectively extracting IP-10 and removing high-abundance serum proteins through acidified protein precipitation (PPT). It was revealed in this work that these high-abundance serum proteins, if not separated from the protein of interest, could cause significant ionization saturation and high background noise in selected reaction monitoring (SRM), leading to a 100-fold higher lower limit of quantification (LLOQ). RESULTS: Our method demonstrated that the acidified PPT could be optimized to selectively extract the protein of interest with full recovery of 97% to 103%, while the high-abundance serum proteins could be effectively removed with minimal matrix effect of 90% to 93%. For the first time, a highly sensitive LC/MS method with a LLOQ of 31.62 pM for the quantitation of IP-10 has been achieved, which is a 100-fold improvement over the generic method. CONCLUSIONS: The described method offers excellent sensitivity with advantages of being antibody reagent independent and leads to significant cost and time savings. It has been successfully employed to determine both total and free IP-10 levels in human serum samples. This method development strategy may also be applied to other small proteins.


Assuntos
Quimiocina CXCL10/sangue , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Proteínas Sanguíneas/química , Quimiocina CXCL10/química , Formiatos , Humanos , Análise dos Mínimos Quadrados , Sensibilidade e Especificidade , Tripsina
20.
Anal Biochem ; 452: 10-2, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24534252

RESUMO

L-serine-O-phosphate (L-SOP), the precursor of L-serine, is a potent agonist against the group III metabotropic glutamate receptors (mGluRs) and, thus, is of interest as a potential biomarker for monitoring modulation of neurotransmitter release. So far, no reports are available on the analysis of L-SOP in cerebrospinal fluid (CSF). Here a novel method is presented to determine L-SOP levels in CSF employing precolumn derivatization with (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) coupled to liquid chromatography/mass spectrometry (derivatization-LC/MS, d-LC/MS).


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Fosfosserina/líquido cefalorraquidiano , Fosfosserina/química , Compostos Organofosforados/química , Succinimidas/química
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