RESUMEN
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.
Asunto(s)
Cromatografía Liquida/métodos , Inmunoensayo/métodos , Espectrometría de Masas en Tándem/métodos , Bioensayo , Biomarcadores/análisis , Humanos , Proteínas/análisis , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: A previously described immunoaffinity (IA)-LC-MS/MS assay for human ß-nerve growth factor (ß-NGF) was implemented to support large-scale sample testing for multiple clinical trials. Methodology & results: The procedure was modified to increase throughput by simultaneous preparation of two 96-well plates and LC duty-cycle reduction. Robustness of the LC method and nano-ESI was ensured during large-scale assay execution by closely monitoring and, if needed, replacing system components prior to failure. Following validation, the assay was used to analyze approximately 19,000 samples from multiple clinical studies over several years. CONCLUSION: Routine implementation of the ß-NGF IA-LC-MS/MS assay supported drug development programs. This optimized assay format now serves as a template for other clinical protein biomarker assays.
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Cromatografía Líquida de Alta Presión , Factor de Crecimiento Nervioso/análisis , Espectrometría de Masas en Tándem , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión/normas , Humanos , Nanotecnología/normas , Factor de Crecimiento Nervioso/aislamiento & purificación , Factor de Crecimiento Nervioso/normas , Control de Calidad , Proteínas Recombinantes/análisis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/normas , Espectrometría de Masas en Tándem/normasRESUMEN
This paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group's Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion. Within this context, we considered a range of sample preparation approaches from simple in-matrix protein denaturation and digestion to complex procedures involving affinity capture enrichment. Consideration was given to the method validation experiments normally associated with traditional LC-MS/MS and ligand-binding assays. Our collective experience, thus far, is that LC-MS/MS methods for protein bioanalysis require different development and validation considerations than those used for small molecules. The method development and validation plans need to be tailored to the particular assay format being established, taking into account a number of important factors: the intended use of the assay, the test species or study population, the characteristics of the protein biotherapeutic and its similarity to endogenous proteins, potential interferences, as well as the nature, quality, and availability of reference and internal standard materials.
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Cromatografía Liquida/métodos , Proteínas/análisis , Espectrometría de Masas en Tándem/métodos , Animales , Canadá , Humanos , Estados Unidos , Estudios de Validación como AsuntoRESUMEN
The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for Large molecules bioanalysis using LBA and Immunogenicity. Part 1 (Small molecules bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input) were published in the Bioanalysis issues 6(22) and 6(23), respectively.
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Técnicas de Química Analítica , Inmunidad , Anticuerpos Neutralizantes/inmunología , Biotransformación , Humanos , Preparaciones Farmacéuticas/metabolismo , Farmacocinética , Polietileno/química , Guías de Práctica Clínica como Asunto , Estados Unidos , United States Food and Drug AdministrationRESUMEN
The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.
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Técnicas de Química Analítica/normas , Guías como Asunto , Estudios de Validación como Asunto , Biomarcadores/análisis , Calibración , Ligandos , Límite de Detección , Juego de Reactivos para Diagnóstico , Reproducibilidad de los Resultados , Estados Unidos , United States Food and Drug AdministrationRESUMEN
The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' input) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the upcoming issues of Bioanalysis.
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Bioensayo , Cromatografía Liquida/métodos , Espectrometría de Masas/métodosRESUMEN
The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input. Part 1 (Small molecules bioanalysis using LCMS) was published in the Bioanalysis issue 6(22) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the Bioanalysis issue 6(24).
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Técnicas de Laboratorio Clínico , Métodos Analíticos de la Preparación de la Muestra , Cromatografía Liquida , Humanos , Espectrometría de MasasRESUMEN
The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These 'hot' topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science. Such as the previous editions, this 2013 White Paper addresses important bioanalytical issues and provides practical answers to the topics presented, discussed and agreed upon by the global bioanalytical community attending the 7th Workshop on Recent Issues in Bioanalysis.
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Descubrimiento de Drogas/métodos , Animales , Bioquímica/métodos , Bioquímica/normas , Biomarcadores Farmacológicos/análisis , California , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Aprobación de Drogas/métodos , Descubrimiento de Drogas/normas , Humanos , Farmacocinética , Estudios de Validación como AsuntoRESUMEN
Intracranial dural arteriovenous fistulas are abnormal communications between higher-pressure arterial circulation and lower-pressure venous circulation. This abnormal communication can result in important and frequently misdiagnosed neurological abnormalities.A case of rapid onset paraplegia following cervical chiropractic manipulation is reviewed. The patient's generalized spinal cord edema, lower extremity paraplegia and upper extremity weakness, were initially believed to be a complication of the cervical spinal manipulation that had occurred earlier on the day of admission. Subsequent diagnostic testing determined the patient suffered from impaired circulation of the cervical spinal cord produced by a Type V intracranial arteriovenous fistula and resultant venous hypertension in the pontomesencephalic and anterior spinal veins.The clinical and imaging findings of an intracranial dural arteriovenous fistula with pontomesencephalic venous congestion and paraplegia are reviewed.This case report emphasizes the importance of thorough and serial diagnostic imaging in the presence of sudden onset paraplegia and the potential for error when concluding atypical neurological presentations are the result of therapeutic misadventure.
RESUMEN
BACKGROUND: Quantitation of biomarkers by LC-MS/MS is complicated by the presence of endogenous analytes. This challenge is most commonly overcome by calibration using an authentic standard spiked into a surrogate matrix devoid of the target analyte. A second approach involves use of a stable-isotope-labeled standard as a surrogate analyte to allow calibration in the actual biological matrix. For both methods, parallelism between calibration standards and the target analyte in biological matrix must be demonstrated in order to ensure accurate quantitation. RESULTS: In this communication, the surrogate matrix and surrogate analyte approaches are compared for the analysis of five amino acids in human plasma: alanine, valine, methionine, leucine and isoleucine. In addition, methodology based on standard addition is introduced, which enables a robust examination of parallelism in both surrogate analyte and surrogate matrix methods prior to formal validation. Results from additional assays are presented to introduce the standard-addition methodology and to highlight the strengths and weaknesses of each approach. CONCLUSION: For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.
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Aminoácidos/sangre , Cromatografía Liquida , Espectrometría de Masas en Tándem , Aminoácidos/normas , Ácidos Araquidónicos/sangre , Ácidos Araquidónicos/normas , Biomarcadores/análisis , Cromatografía Liquida/normas , Endocannabinoides/sangre , Endocannabinoides/normas , Glicéridos/sangre , Glicéridos/normas , Humanos , Marcaje Isotópico , Metilhistidinas/sangre , Alcamidas Poliinsaturadas/sangre , Alcamidas Poliinsaturadas/normas , Estándares de Referencia , Espectrometría de Masas en Tándem/normasRESUMEN
In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (>104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.
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Antimetabolitos Antineoplásicos/análisis , Cromatografía Líquida de Alta Presión/métodos , ADN/química , Desoxicitidina/análogos & derivados , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral , Desoxicitidina/análisis , Desoxicitidina/farmacología , Desoxiguanosina/química , Perros , Humanos , GemcitabinaRESUMEN
Liquid chromatography combined with electrospray ionization is widely used for direct analysis of polar and labile molecules by LCMS. The on-line coupling in LCMS is a major strength but also causes a principal limitation that each eluting analyte has to be analyzed immediately and is not available for detailed interrogation after the LCMS run. Here we developed a new chromatographic strategy, which removes this limitation. After column separation the flow is split, one portion is analyzed directly, and the other is diverted to a capture capillary. After the direct LCMS run, the flow is switched, and the portion stored in the capillary is analyzed ("replay run"). We describe a setup consisting of an analytical column, a splitting valve, and a focusing column, which performs at full sensitivity and undiminished chromatographic resolution. We demonstrate three principal advantages of this system: nearly continuous MS utilization, duplicate analysis without requirement for additional sample, and targeting of important but undersampled features in the replay run.
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Cromatografía Liquida/métodos , Hígado/química , Proteoma/análisis , Espectrometría de Masas en Tándem , Animales , Ratones , Sensibilidad y Especificidad , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismoRESUMEN
Some mutations arise in association with a potential sequence donor that consists of an imperfect direct or reverse repeat. Many such mutations are complex; that is, they consist of multiple close sequence changes. Current models posit that the primer terminus of a replicating DNA molecule dissociates, reanneals with an ectopic template, extends briefly, and then returns to the cognate template, bringing with it a locally different sequence; alternatively, a hairpin structure may form the mutational intermediate when processed by mismatch repair. This process resembles replication repair, in which primer extension is blocked by a lesion in the template; in this case, the ectopic template is the other daughter strand, and the result is error-free bypass of the lesion. We previously showed that mutations that impair replication repair can enhance templated mutagenesis. We show here that the intensity of templated mutation can be exquisitely sensitive to its local sequence, that the donor and recipient arms of an imperfect inverse repeat can exchange roles, and that double mutants carrying two alleles, each affecting both templated mutagenesis and replication repair, can have unexpected phenotypes. We also record an instance in which the mutation rates at two particular sites change concordantly with a distant sequence change, but in a manner that appears unrelated to templated mutagenesis.
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Bacteriófago T4/genética , Mutación , Moldes Genéticos , Secuencia de Bases , Codón/genética , Análisis Mutacional de ADN , Cartilla de ADN , Replicación del ADN , ADN Viral/genética , Escherichia coli/genética , MutagénesisRESUMEN
BACKGROUND CONTEXT: Persistence of a primary sacral ossification center resulting in synchondrosis in adulthood is rare and can confound diagnostic decision making during patient management. PURPOSE: To present a synchondrosis between the sacral ala and sacral body in a healthy 23-year-old US Marine who had low back pain. STUDY DESIGN/SETTING: Case report. OUTCOME MEASURES: Self-report measures included a numerical pain scale and Roland Morris Disability questionnaire; physiological measures included plain film radiography, computed tomography scans, magnetic resonance imaging, and physical examination procedures; and functional measures included the patient's ability to run and sit without pain and to maintain US Marine Corps fitness standards. METHODS: The initial management of his low back pain included a course of nonsteroidal anti-inflammatory medication, chiropractic manipulation of the sacroiliac joints and adjacent tissues, and therapeutic exercise. When the patient's condition did not improve as quickly as anticipated, plain X-ray films were ordered; this revealed a vertical cleft in the sacrum at the site of the patient's pain. Further imaging showed the anomalous cleft to be a synchondrosis between the costal element and the centrum of the sacrum. Manual manipulation, physical training, and ergonomic advice were continued. RESULTS: Pain severity decreased from 7 to 0, and the Roland Morris score decreased from 14 to 1. He could sit for prolonged periods of time and exercise to Marine Corps standards. CONCLUSIONS: It is unlikely that the synchondrosis was the structure responsible for generating the patient's low back pain. However, such an anomaly is clinically relevant because it may mimic a fracture.
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Dolor de la Región Lumbar/patología , Osificación Heterotópica/patología , Sacro/patología , Adulto , Humanos , Dolor de la Región Lumbar/diagnóstico por imagen , Dolor de la Región Lumbar/terapia , Imagen por Resonancia Magnética , Masculino , Osificación Heterotópica/diagnóstico por imagen , Osificación Heterotópica/terapia , Sacro/diagnóstico por imagen , Tomografía Computarizada por Rayos XRESUMEN
Replication repair mediates error-free bypass of DNA damage in a series of steps that include regression of the replication fork, primer-terminus switching to use the other daughter strand as an undamaged template, primer extension, primer switching back to its cognate template with the primer terminus now having bypassed the damage, and fork rearrangement to a normal configuration. By both genetic and biochemical criteria, bacteriophage T4 catalyzes replication repair with two alternative sets of proteins, one including the gp32 SSB and the gp41 DNA helicase and the other including the UvsX recombinase. In each pathway, synthesis is conducted by the gp43 DNA polymerase. Here we show that defects in gp32, gp41 or UvsX that impair replication repair also increase mutation rates generally, but especially for templated mutations. Such templated mutations are associated with palindromic or direct repeats that are either perfect or imperfect. Models of templated mutagenesis require that the primer terminus switches to an ectopic template, but one that yields mutations instead of error-free bypass. We suggest that the proteins that conduct replication repair normally direct a blocked primer strand specifically to the other daughter strand with considerable accuracy, but that strand switching becomes promiscuous when these proteins are mutationally impaired, thus promoting templated mutations.
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Reparación del ADN , Replicación del ADN , Mutación , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Secuencia de Bases , Daño del ADN , ADN Viral/genética , ADN Viral/metabolismo , Escherichia coli/virología , Genes Virales , Modelos Genéticos , Datos de Secuencia MolecularRESUMEN
Whenever the name Edmund Husserl appears in the context of nursing research, what correctly comes to mind is the phenomenological approach to qualitative methodology. Husserl is not only considered the founder of phenomenology, but his broad concept development also contributed to the demise of positivism and inspired fruitful approaches to the social sciences. In this spirit of inspiration, it must be expressed that Husserl's theory of wholes and parts, and particularly his differentiation of parts into "pieces" and "moments", is very helpful in guiding the selection of research methods across the board in nursing science. The purpose of this paper is to highlight a frame of reference for nursing researchers to use in examining the essential nature of that which is being studied. This frame of reference is the Husserlian philosophy of "pieces" and "moments" in relation to the whole. "Pieces" are independent parts of the whole that are subject to isolability in study, whereas "moments" are nonindependent parts, which cannot be detached, presented, or studied apart from the whole. The intent is to propose this frame of reference as a philosophical base from which nursing researchers may better select among paradigms and methodological approaches in regard to the essential nature ("pieces" or "moments") of that which they are researching.
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Investigación en Enfermería/métodos , Filosofía en Enfermería/historia , Historia del Siglo XX , Humanos , Investigación en Enfermería/historia , Investigación CualitativaRESUMEN
Quantitative bioanalysis by direct nanoelectrospray infusion coupled to tandem mass spectrometry has been achieved using an automated liquid sampler integrated with an array of microfabricated electrospray nozzles allowing rapid, serial sample introduction (1 min/ sample). Standard curves prepared in human plasma for verapamil (r2 = 0.999) and its metabolite norverapamil (r2 = 0.998) were linear over a range of 2.5-500 ng/ mL. Based on the observed precision and accuracy, a lower limit of quantitation of 5 ng/mL was assigned for both analytes. Sample preparation consisted of protein precipitation with an organic solvent containing the structural analogue gallopamil as an internal standard. Protein precipitation was selected both to maximize throughput and to test the robustness of direct nanoelectrospray infusion. Aliquots of supernatant (10 pL) were transferred to the back plane of the chip using disposable, conductive pipet tips for direct infusion at a flow rate of 300 nL/min. Electrospray ionization occurred from the etched nozzles (30-microm o.d.) on the front of the chip, initiated by a voltage applied to the liquid through the pipet tip. The chip was positioned near the API sampling orifice of a triple quadrupole mass spectrometer, which was operated in selected reaction monitoring mode. Results are presented that document the complete elimination of system carry-over, attributed to lack of a redundant fluid path. This technology offers potential advantages for MS-based screening applications in drug discovery by reducing the time for methods development and sample analysis.
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Monitoreo de Drogas/métodos , Espectrometría de Masa por Ionización de Electrospray , Verapamilo/análogos & derivados , Verapamilo/sangre , Humanos , Preparaciones Farmacéuticas/sangre , Estándares de Referencia , Verapamilo/metabolismoRESUMEN
Single nucleotide polymorphisms (SNPs) are currently being mapped and databased at a remarkable pace, providing a viable means for understanding disease susceptibility, differential drug response and human evolution. Consequently, there is an increasing demand for SNP genotyping technologies that are simple, rapid, cost effective and readily amenable to automation for high-throughput analyses. In this study, we improved the Survivor Assay, a SNP detection method based on electrospray ionization mass spectrometry (ESI-MS), with several developments. One improvement is the development of a one-well assay, requiring no off-line purification of the polymerase chain reaction product, achieved by simple addition of reagent solution into a single well. Another is the on-line separation of magnesium and dideoxynucleotides using an in-house made monolithic metal chelating column, eliminating any off-line sample preparation prior to mass spectrometric analysis. Here the Survivor Assay is extended from a proof-of-principle concept to a validated method by genotyping six SNPs from five different regions of human genomic DNA in 55 individual samples with 100% accuracy. This improved Survivor Assay eliminates the tedious and time-consuming steps of sample preparation, minimizes sample handing and offers a high-throughput analysis of SNPs by ESI-MS. The current combined preparation and analysis time is 2 min per sample. The simplicity of this method has potential for full automation and parallel chromatography and, thus, reduced analysis time. In addition, we have adapted the Survivor Assay for quantitative SNP analysis in pooled DNA samples. The capabilities and sensitivity of this approach were evaluated. We demonstrate that an allele occurring at a frequency of 2% can consistently be quantitated.
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Polimorfismo de Nucleótido Simple/genética , Alelos , Cromatografía de Afinidad , ADN/análisis , ADN/química , ADN/genética , Cartilla de ADN/química , Genotipo , Humanos , Oligonucleótidos/química , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Macroporous poly(styrene-divinylbenzene) (PS-DVB) monoliths were prepared by in situ polymerization in PEEK, fused silica, or stainless steel tubing having an inner diameter of 75 or 125 microm. A process is described for subsequent alkylation of the flow-contacting surfaces of the monoliths. The process treats all the surfaces including through-pore surfaces of the rigid macroporous monolith with a solution containing a dissolved Friedel-Crafts catalyst, an alkyl halide (1-chlorooctadecane), and an organic solvent. This process produces an improved reversed-phase liquid chromatographic separation of peptides compared to an unmodified monolithic PS-DVB column. The surface octadecylation is not necessary for a reversed-phase separation of proteins since both unmodified and modified columns provide comparable results. Tryptic protein digests, standard proteins, and standard peptides were used to evaluate the monolithic columns by employing electrospray mass spectrometry detection. Potential applications in proteomics studies by mass spectrometry, which use the alkylated monolithic column engaged onto the nanofabricated electrospray ionization chip, are also discussed.