RESUMEN
Insulin degludec is an ultra-long-acting insulin analogue that is increasingly being used in diabetes due to its favourable efficacy and safety profile. Thus, there is an increasing demand for a reliable and specific analytical method to quantify insulin degludec for research, pharmaceutical industry and clinical applications. We developed and validated an automated, high-throughput method for quantification of insulin degludec in human blood samples across the expected clinical range combining immunopurification with high-resolution mass spectrometry. Validation was performed according to the requirements of the US Food and Drug Administration. The method satisfyingly met the following parameters: lower limit of quantification (120 pM), linearity, accuracy (error < 5%), precision (CV < 7.7%), selectivity, carry-over, recovery (89.7-97.2%), stability and performance in the presence of other insulin analogues. The method was successfully applied to clinical samples of patients treated with insulin degludec showing a good correlation with the administered dose (r2 = 0.78). High usability of the method is supported by the small specimen volume, automated sample processing and short analysis time. In conclusion, this reliable, easy-to-use and specific mass spectrometric insulin degludec assay offers great promise to address the current unmet need for standardized insulin analytics in academic and industrial research. Graphical Abstract.
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Cromatografía Liquida/métodos , Insulina de Acción Prolongada/análisis , Espectrometría de Masas/métodos , Adulto , Humanos , Insulina de Acción Prolongada/aislamiento & purificación , Límite de Detección , Masculino , Reproducibilidad de los ResultadosRESUMEN
The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi-automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.
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Cromatografía Liquida/métodos , Ensayos Analíticos de Alto Rendimiento , Inmunoensayo/métodos , Insulina/análogos & derivados , Insulina/sangre , Proteómica , Espectrometría de Masas en Tándem/métodos , Humanos , Isoformas de ProteínasRESUMEN
Measurement of insulin and its therapeutic analogs is important in diabetes, hypoglycemia, sports anti-doping and toxicology. Commercial insulin immunoassays fail to detect commonly prescribed insulin analogs. Because of their unique sequences and masses, these analogs are readily measured and distinguished with mass spectrometric (MS) assays. Reviewed here is an insulin mass spectrometric immunoassay (MSIA) that combines micro-scale immunoaffinity capture with sensitive MS detection of insulin and its therapeutic analogs. An antibody reactive to all insulin analogs was used to affinity capture the insulin analogs. Following elution, insulins were detected with MALDI-TOF MS or LC-MS analysis. Isotopic resolution for insulin was achieved for both MS techniques, and several insulin analogs were detected at unique m/z signals. Porcine insulin, spiked in all samples, served as an internal reference standard for quantification. Linear standard curves spanning three orders of magnitude were obtained, with limits of detection of 15pM for the MALDI-TOF MS and 1.5pM for the LC-MS. This insulin assay was capable of detecting and quantifying not only human endogenous insulin, but also most of the therapeutic insulin analogs, which could find use in diagnosis of severe hypoglycemia and in sports anti-doping. SIGNIFICANCE: Insulin replacement therapy consists of injection of long- or fast-acting insulin analogs with slightly modified primary sequences compared to human insulin. Assays that are capable of detecting all insulin analogs are desired, not only for medical management of diabetes and severe hypoglycemia but also for sports anti-doping and toxicology.
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Inmunoensayo/métodos , Insulina/análisis , Espectrometría de Masas/métodos , Animales , Anticuerpos , Biología Computacional , Diabetes Mellitus/tratamiento farmacológico , Doping en los Deportes , Humanos , Hipoglucemia/tratamiento farmacológico , Insulina/análogos & derivados , Insulina/normas , Insulina Regular Porcina/análisis , Multimerización de Proteína , PorcinosRESUMEN
Affinity mass spectrometry is a proteomics approach for selectively isolating target proteins from complex biological fluids for mass spectrometric analysis. When executed in high throughput mode through affinity pipets, the resulting volumetric mass spectrometry arrays enable rapid protein assaying from hundreds of samples. Furthermore, in combination with postcapture proteolytic degradation, this top-down proteomics approach can reveal structural features (i.e., modifications) in the protein sequences that are result of posttranslational modifications and/or point mutations. Described here in greater detail are the individual steps of the high throughput combination of affinity protein capture in antibody-derivatized affinity pipets, protein elution, and protein processing through enzyme-derivatized mass spectrometry targets.
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Fragmentos de Péptidos/análisis , Análisis por Matrices de Proteínas/métodos , Proteínas/análisis , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
Affinity mass spectrometry (AMS) is a proteomics approach for selectively isolating target protein(s) from complex biological fluids for mass spectrometric analysis. The resulting high-content mass spectrometry (MS) data show the unique MS protein signatures (wild-type, posttranslationally modified, as well as genetically modified forms of the protein target) that are present within a biological sample. Information regarding such protein diversity is normally lost in classical proteomic or immunoassay analyses. This chapter presents a step-by-step description of high-throughput AMS in the population proteomic screening of the human plasma protein cystatin C.
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Espectrometría de Masas/métodos , Secuencia de Aminoácidos , Automatización , Proteínas Sanguíneas/química , Calibración , Cistatina C , Cistatinas/sangre , Cistatinas/química , Humanos , Inmunoensayo/métodos , Datos de Secuencia Molecular , Mutación Puntual , Procesamiento Proteico-Postraduccional , Proteínas/química , Proteómica/métodos , Robótica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Activation of cyclic nucleotide-dependent signaling pathways leads to phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)-PTD or FITC-PTD-HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD-phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.
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Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/fisiología , Músculo Liso Vascular/fisiología , Fosfoproteínas/química , Fosfoproteínas/fisiología , Vasodilatación , Secuencias de Aminoácidos , Animales , Línea Celular , Técnicas de Cultivo , Proteínas del Choque Térmico HSP20 , Proteínas de Choque Térmico/metabolismo , Modelos Biológicos , Músculo Liso Vascular/efectos de los fármacos , Miocardio/citología , Nucleótidos Cíclicos/metabolismo , Papaverina/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Fosfopéptidos/genética , Fosfoproteínas/metabolismo , Fosforilación , Conejos , Serotonina/farmacología , Porcinos , Transducción Genética , VasoconstricciónRESUMEN
Insulin like growth factor (IGF)-1 and IGF-2 were assayed from human plasma via biomolecular interaction analysis mass spectrometry, utilizing antibodies as ligands for affinity retrieval. Detection of both targeted and non-targeted IGFs in the mass spectra indicated possible protein complex retrieval by the individual antibodies. A series of control experiments eliminated the possibility of analyte cross-walking between flow cells, significant antibodies cross-reactivity, and direct IGF interactions. To disrupt the putative protein complex and release its constituent proteins, plasma samples were treated with detergents. An SDS-treated plasma yielded IGF signals in a different ratio than the one observed in the mass spectra from the non-treated plasma, suggesting disruption of the protein complex, and its retrieval from non-treated plasma. Novel truncated IGF-2 variant, missing its N-terminal Alanine, was detected in all mass spectra.
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Factor II del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Resonancia por Plasmón de Superficie/métodos , Humanos , Factor I del Crecimiento Similar a la Insulina/inmunología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/inmunología , Factor II del Crecimiento Similar a la Insulina/metabolismo , LigandosRESUMEN
Serum amyloid A protein (SAA) is a human plasma protein that has been recognized as potential biomarker of multiple ailments including myocardial infarction, inflammatory disease and amyloiosis. Presented here is the application of a novel immunoassay technique, termed mass spectrometric immunoassay for the detection and identification of SAA present in human plasma. Results demonstrate the ability to readily detect known SAA isotypes, and to identify novel truncated forms of SAA, in the plasma of healthy individuals and those suffering from acute and chronic inflammation. The approach represents a rapid and sensitive means for the routine structural characterization of known SAA isotypes and the discovery of associated post-translational modifications.
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Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismo , Secuencia de Aminoácidos , Humanos , Masculino , Oligopéptidos/química , Fragmentos de Péptidos/química , Eliminación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.
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Inmunoensayo/métodos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Espectrometría de Masas/métodos , Biomarcadores/sangre , Humanos , Inmunoensayo/normas , Factor I del Crecimiento Similar a la Insulina/química , Espectrometría de Masas/normas , Estándares de Referencia , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.
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Proteínas Sanguíneas/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Inmunoensayo/métodos , Espectrometría de Masas/métodos , Enfermedad de Alzheimer/sangre , Enfermedades Cardiovasculares/sangre , Trastornos del Crecimiento/sangre , Humanos , Neoplasias/sangre , Insuficiencia Renal/sangreAsunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Gestacional/tratamiento farmacológico , Hipoglucemiantes/farmacocinética , Insulina de Acción Prolongada/farmacocinética , Placenta/metabolismo , Embarazo en Diabéticas/tratamiento farmacológico , Femenino , Humanos , Insulina Detemir , Placenta/efectos de los fármacos , EmbarazoRESUMEN
The urine proteome (urineome) has become an ideal biological fluid for biomarker study and detection due to its rich protein content as well as its ease and abundance in collection. Protein variation in human plasma has been linked to the presence of disease states in humans; however, it stands to reason that the same is true of the equally complex urineome. In this manuscript we present the combination of two proteomics technologies, mass spectrometric immunoassay and a bioreactive probe, for the detection and characterization of the protein variants of transthyretin (TTR) found in human urine. Coupling these two technologies we could precisely identify protein variants within a population of normal individuals. This is the first report of the detailed characterization of urinary TTR using this combination of proteomic analytical techniques, and demonstrates a novel non-invasive methodology for the routine analysis of this clinically significant urine protein target.
RESUMEN
BACKGROUND: The myocardium secretes B-type natriuretic peptide (BNP) in response to stimuli associated with heart failure (HF). However, high immunoreactive-BNP levels in patients with HF are associated with a paradoxical lack of natriuretic response. We hypothesized that commercially available assays for immunoreactive BNP do not reflect the bioactivity of the natriuretic peptide system, because they measure both unprocessed inactive pro-BNP and mature BNP 1-32. We describe an assay for the detection of bioactive BNP 1-32 and confirm very low concentrations in plasma from HF patients. METHODS AND RESULTS: We developed a quantitative mass spectrometry immunoassay to capture endogenous BNP peptides using high affinity antibodies. Bound BNP and its truncated fragments were detected by matrix assisted laser desorption ionization-time of flight mass spectrometry based on their predicted masses. Mass spectrometry immunoassay revealed rapid in vitro degradation of BNP 1-32 in plasma, which requires plasma collection in the presence of high protease inhibitor concentrations. In 11 of 12 HF patients BNP 1-32 was detectable, ranging from 25 to 43 pg/mL. Several degraded forms of BNP were also detected at similarly low levels. In contrast, parallel measurements of immunoreactive BNP using the Biosite assay ranged from 900 to 5000 pg/mL. CONCLUSIONS: Detection of endogenous BNP 1-32 requires special preservation of plasma samples. Mass spectrometry immunoassay technology demonstrates that HF patients have low levels of BNP 1-32. Commercially available immunoreactive-BNP assays overrepresent biological activity of the natriuretic peptide system because they cannot distinguish between active and inactive forms. This observation may, in part, explain the "natriuretic paradox."
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Insuficiencia Cardíaca/sangre , Inmunoensayo/métodos , Espectrometría de Masas/métodos , Péptido Natriurético Encefálico/sangre , Anciano , Anciano de 80 o más Años , Conservación de la Sangre/métodos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana Edad , Péptido Natriurético Encefálico/efectos de los fármacos , Péptido Natriurético Encefálico/metabolismo , Concentración Osmolar , Inhibidores de Proteasas/administración & dosificaciónRESUMEN
This review outlines the concept of population proteomics and its implication in the discovery and validation of cancer-specific protein modulations. Population proteomics is an applied subdiscipline of proteomics engaging in the investigation of human proteins across and within populations to define and better understand protein diversity. Population proteomics focuses on interrogation of specific proteins from large number of individuals, utilizing top-down, targeted affinity mass spectrometry approaches to probe protein modifications. Deglycosylation, sequence truncations, side-chain residue modifications, and other modifications have been reported for myriad of proteins, yet little is know about their incidence rate in the general population. Such information can be gathered via population proteomics and would greatly aid the biomarker discovery efforts. Discovery of novel protein modifications is also expected from such large scale population proteomics, expanding the protein knowledge database. In regard to cancer protein biomarkers, their validation via population proteomics-based approaches is advantageous as mass spectrometry detection is used both in the discovery and validation process, which is essential for the detection of those structurally modified protein biomarkers.
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Biomarcadores de Tumor/metabolismo , Genética de Población/métodos , Proteómica/métodos , Biomarcadores de Tumor/química , Humanos , Espectrometría de Masas , Reproducibilidad de los ResultadosRESUMEN
This report addresses the need for additional assays for human resistin (hRES) by developing a rational progression of the mass spectrometric immunoassay to incorporate recombinant proteins. The recombinant-based hRES mass spectrometric immunoassay (RES-MSIA) was initially developed for the qualitative analysis of the human resistin homodimer from normal (healthy) plasma samples. The method involved selective extraction and detection of both endogenous and recombinant resistant proteins. RES-MSIA was then applied to the rigorous quantification of resistin. The resistin standard addition curve was constructed from serially diluted concentrations of rhRES using endogenous hRES, inherent in the human plasma, as the internal reference standard (IRS). The roles of endogenous and recombinant resistin were subsequently reversed, using rhRES as the IRS during RES-MSIA quantification. Concurrently, the relative ratio of hRES to rhRES was used as an ancillary technique to rapidly determine the relative concentration of hRES in each of plasma samples. Overall, normal hRES levels determined by RES-MSIA were found to be comparable to those selected and determined by ELISA. With regard to gender, female donor samples were slightly elevated over males. Four single cardiac samples were analyzed and found to have hRES concentrations approximately three times that of the normal. The recombinant-based RES-MSIA is rapid and is amendable to parallel high-throughput robotic processing of resistin related disease cohorts.
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Resistina/sangre , Resistina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Perfilación de la Expresión Génica , Humanos , Inmunoensayo , Análisis por Matrices de Proteínas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/químicaRESUMEN
We report a new DNA sequencing-by-synthesis method in which the sequences of DNA templates, hybridized to a surface-immobilized array of DNA primers, are determined by sensing the number of nucleotides by which the primers in each array spot are extended in sequential DNA polymerase-catalyzed nucleotide incorporation reactions, each with a single fluorescein-labeled deoxyribonucleoside triphosphate (dNTP) species. The fluorescein label is destroyed after each readout by a photostimulated reaction with diphenyliodonium chloride. A DNA polymerase with enhanced ability to incorporate, and to extend beyond, modified nucleotides is used. Self-quenching of adjacent fluorescein labels, which impedes readout of homopolymeric runs, is avoided by diluting the labeled dNTP with unlabeled reagent. Misincorporation effects have been quantified and are small; however, low-level contamination of dNTPs with other nucleotides mimics misincorporation and can produce significant false-positive signals. These impurities are removed by polymerase-catalyzed incorporation into complementary "cleaning duplexes." Here, we demonstrate the accurate sequence readout for a small array of known DNA templates, the ability to quantify homopolymeric runs, and a short sequencing example of sections of the wild-type and mutant BRCA1 gene. For a 20,000-spot array, readout rates in excess of 6000 bases per minute are projected.
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Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia de ADN/métodos , Secuencia de Aminoácidos , ADN Polimerasa Dirigida por ADN , Genes BRCA1 , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Plasma proteins represent an important part of the human proteome. Although recent proteomics research efforts focus largely on determining the overall number of proteins circulating in plasma, it is equally important to delineate protein variations among individuals, because they can signal the onset of diseases and be used as biological markers in diagnostics. To date, there has been no systematic proteomics effort to characterize the breadth of structural modifications in individual proteins in the general population. In this work, we have undertaken a population proteomics study to define gene- and protein-level diversity that is encountered in the general population. Twenty-five plasma proteins from a cohort of 96 healthy individuals were investigated through affinity-based mass spectrometric assays. A total of 76 structural forms/variants were observed for the 25 proteins within the samples cohort. Posttranslational modifications were detected in 18 proteins, and point mutations were observed in 4 proteins. The frequency of occurrence of these variations was wide-ranged, with some modifications being observed in only one sample, and others detected in all 96 samples. Even though a relatively small cohort of individuals was investigated, the results from this study illustrate the extent of protein diversity in the human population and can be of immediate aid in clinical proteomics/biomarker studies by laying a basal-level statistical foundation from which protein diversity relating to disease can be evaluated.
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Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Adolescente , Adulto , Anciano , Proteínas Sanguíneas/metabolismo , Estudios de Cohortes , Femenino , Marcadores Genéticos , Variación Genética , Genética de Población , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Mutación Puntual , Procesamiento Proteico-Postraduccional , ProteómicaRESUMEN
A high-throughput affinity capture and reduction approach was developed for phenotype and post-translational modification analysis of a complexed globular protein, haptoglobin (Hp), directly from human plasma. Hp was selectively retrieved utilizing anti-Hp antibodies immobilized onto affinity pipette tips, eluted onto a formatted mass spectrometer target for reduction of Hp alpha-chains (Hpalpha1 and Hpalpha2) and subjected to subsequent MALDI-MS analysis. The affinity capture and reduction approach was originally developed from a pre-extraction reduction methodology that was optimized to an affinity capture post-reduction technique for intact Hp alpha-chain variant analysis, phenotype classification and ensuing post-translational variant detection. Three common Hp phenotypes (1-1, 2-1 and 2-2) were assigned according to detection of Hpalpha1 and/or Hpalpha2 reduced intact chain(s) average mass(es). The affinity capture post-reduction approach was scaled for high-throughput Hp alpha-chain phenotype analysis from a normal plasma cohort. The entire sample cohort was successfully analyzed and phenotyped using the developed approach. Additionally, Hp post-translational variants were detected and assigned via accurate MS analyses. The results of this study suggest use of the methodology in future analyses of other similarly complexed proteins and in normal versus disease cohort population proteomics studies.
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Haptoglobinas/química , Espectrometría de Masas/métodos , Fenotipo , Haptoglobinas/genética , Humanos , Inmunoensayo/métodos , Procesamiento Proteico-PostraduccionalRESUMEN
Mass spectrometric immunoassay (MSIA) is a proteomics technology that combines the selectivity of affinity capture with the sensitivity and resolution of mass spectrometric detection. This unique approach allows for intact protein identification therefore is readily capable of discriminating between protein variants, i.e., mutations, posttranslational modifications, and truncations. In this work, MSIA is used in the comparative analyses of retinol binding protein (RBP) from the plasma and urine of a small study population. Detailed RBP profiles were obtained from both biological fluids, resulting in the identification of several catabolic RBP products (present in urine) that have not been previously reported. In addition, comparative analysis of urine samples taken from healthy and renally impaired individuals revealed different breakdown profiles. These results illustrate the use of MSIA for the rapid, sensitive, and accurate profiling of RBP both within and between individuals.
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Fenotipo , Proteínas de Unión al Retinol/metabolismo , Proteínas de Unión al Retinol/orina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Isoformas de Proteínas , Pielonefritis/sangre , Pielonefritis/orina , Proteínas Plasmáticas de Unión al RetinolRESUMEN
Serum amyloid P component (SAP) is a human plasma protein that has been widely studied for its influence on amyloid plaque formation and stabilization. SAP was characterized directly from human plasma and urine samples via novel affinity mass spectrometry-based proteomic technology that is able to readily discriminate between mass-altered protein variants. These analyses were able to identify several variants of SAP that have not been previously reported. These variants include microheterogeneity of the glycan structure, from the loss of one or both terminal sialic acid residues, as well as the loss of the C-terminal valine residue. Moreover, the analysis of urine allowed for the consistent identification of serum amyloid P component as a normal constituent of the urine proteome.