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INTRODUCTION: We explored the variations of blood biomarkers of Alzheimer's disease (AD) by chronic diseases and systemic inflammation. METHODS: We explored the association of AD blood biomarkers with chronic diseases and systemic inflammation (interleukin-6 [IL-6]), in 2366 dementia-free participants of the Swedish National Study on Aging and Care-in Kungsholmen, using quantile regression models. RESULTS: A greater number of co-occurring chronic diseases was associated with higher concentrations of phosphorylated-tau 181 (p-tau181), total-tau (t-tau), neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP) (p < 0.01). Anemia, kidney, cerebrovascular, and heart diseases were associated with variations in the levels of AD blood biomarkers. Participants in the highest (vs. lowest) interleukin-6 (IL-6) tertile had higher NfL concentration. Systemic inflammation amplified the associations between several chronic diseases and p-tau181, t-tau, NfL, and GFAP. DISCUSSION: In the community, the concentration of AD blood biomarkers varies in relation to medical conditions and systemic inflammation. Recognizing these influences is crucial for the accurate interpretation and clinical implementation of blood biomarkers. HIGHLIGHTS: Participants with a complex clinical profile (i.e., multiple co-occurring diseases or specific disease combinations) display elevated levels of AD blood-biomarkers. Anemia, heart, cerebrovascular, and kidney diseases are associated with variations is the levels of AD blood biomarkers in cognitively intact older adults. Systemic inflammation amplifies the association between several chronic diseases and AD blood biomarkers.
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Enfermedad de Alzheimer , Biomarcadores , Inflamación , Interleucina-6 , Proteínas tau , Humanos , Enfermedad de Alzheimer/sangre , Biomarcadores/sangre , Femenino , Masculino , Inflamación/sangre , Anciano , Enfermedad Crónica , Suecia/epidemiología , Interleucina-6/sangre , Proteínas tau/sangre , Anciano de 80 o más Años , Proteína Ácida Fibrilar de la Glía/sangre , Proteínas de Neurofilamentos/sangreRESUMEN
Patient-centric sampling strategies, where the patient performs self-sampling and ships the sample to a centralized laboratory for readout, are on the verge of widespread adaptation. However, the key to a successful patient-centric workflow is user-friendliness, with few noncritical user interactions, and simple, ideally biohazard-free shipment. Here, we present a capillary-driven microfluidic device designed to perform the critical biomarker capturing step of a multiplexed immunoassay at the time of sample collection. On-chip sample drying enables biohazard-free shipment and allows us to make use of advanced analytics of specialized laboratories that offer the needed analytical sensitivity, reliability, and affordability. Using C-Reactive Protein, MCP1, S100B, IGFBP1, and IL6 as model blood biomarkers, we demonstrate the multiplexing capability and applicability of the device to a patient-centric workflow. The presented quantification of a biomarker panel opens up new possibilities for e-doctor and e-health applications.
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Laboratorios , Técnicas Analíticas Microfluídicas , Humanos , Reproducibilidad de los Resultados , Inmunoensayo , Biomarcadores , Dispositivos Laboratorio en un Chip , Atención Dirigida al PacienteRESUMEN
BACKGROUND: Patients with liver cirrhosis are recommended ultrasonography screening for early detection of hepatocellular carcinoma to increase the chances of curative treatment. However, ultrasonography alone lacks in sensitivity. Adding plasma biomarkers may increase the detection rate. We performed a broad exploratory analysis to find new plasma proteins with potential applicability for HCC screening in patients with cirrhosis. METHODS: In a protein discovery cohort of 172 patients with cirrhosis or HCC, we screened for 481 proteins with suspension bead array or proximity extension assay. From these, 24 proteins were selected for further analysis in a protein verification cohort (n = 160), using ELISA, Luminex or an electrochemiluminescence platform. A cut-off model and a stepwise logistic regression model were used to find combinations of proteins with the best discriminatory performance between HCC and cirrhosis. RESULTS: Stepwise logistic regression revealed alpha-fetoprotein (AFP), decarboxy-prothrombin (DCP), thioredoxin reductase 1 (TXNRD1), and fibroblast growth factor 21 (FGF21) as the proteins with the best discriminatory performance between HCC and cirrhosis. Adding TXNRD1 to DCP and AFP increased the AUC from 0.844 to 0.878, and combining AFP, DCP and TXNRD1 with age and sex resulted in an AUC of 0.920. FGF21, however, did not further increase the performance when including age and sex. CONCLUSION: In the present study, TXNRD1 improves the sensitivity and specificity of AFP and DCP as HCC screening tools in patients with cirrhosis. We suggest that TXNRD1 should be validated in prospective settings as a new complementary HCC biomarker together with AFP and DCP.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Tiorredoxina Reductasa 1 , Humanos , alfa-Fetoproteínas/análisis , Biomarcadores , Biomarcadores de Tumor , Carcinoma Hepatocelular/diagnóstico por imagen , Cirrosis Hepática/diagnóstico , Neoplasias Hepáticas/diagnóstico por imagen , Estudios Prospectivos , Precursores de Proteínas , Protrombina , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: There is an urgent need for novel blood biomarkers for the detection of Alzheimer's disease (AD). We previously showed that levels of the bisecting N-acetylglucosamine glycan epitope was elevated in cerebrospinal fluid in AD. However, its diagnostic value in blood is unknown. METHODS: We analyzed blood levels of bisecting N-acetylglucosamine and total tau in a retrospective cohort of 233 individuals. Progression to AD was compared between the groups using Cox regression. The predictive value of the biomarkers was determined by logistic regression. RESULTS: Bisecting N-acetylglucosamine correlated with tau levels (p < 0.0001). Individuals with an intermediate tau/bisecting N-acetylglucosamine ratio had elevated AD risk (hazard ratio = 2.06, 95% confidence interval [CI]: 1.18-3.6). Moreover, a combined model including tau/bisecting N-acetylglucosamine ratio, apolipoprotein E (APOE) ε4 status, and Mini-Mental State Examination score predicted future AD (area under the curve = 0.81, 95% CI: 0.68-0.93). DISCUSSION: Bisecting N-acetylglucosamine in combination with tau is a valuable blood biomarker for predicting AD.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Humanos , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/líquido cefalorraquídeo , Apolipoproteína E4/genética , Proteínas tau/líquido cefalorraquídeo , Estudios Retrospectivos , Alelos , Acetilglucosamina , Genotipo , Biomarcadores/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Disfunción Cognitiva/diagnósticoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) refers to excess fat accumulation in the liver. In animal experiments and human kinetic study, we found that administration of combined metabolic activators (CMAs) promotes the oxidation of fat, attenuates the resulting oxidative stress, activates mitochondria, and eventually removes excess fat from the liver. Here, we tested the safety and efficacy of CMA in NAFLD patients in a placebo-controlled 10-week study. We found that CMA significantly decreased hepatic steatosis and levels of aspartate aminotransferase, alanine aminotransferase, uric acid, and creatinine, whereas found no differences on these variables in the placebo group after adjustment for weight loss. By integrating clinical data with plasma metabolomics and inflammatory proteomics as well as oral and gut metagenomic data, we revealed the underlying molecular mechanisms associated with the reduced hepatic fat and inflammation in NAFLD patients and identified the key players involved in the host-microbiome interactions. In conclusion, we showed that CMA can be used to develop a pharmacological treatment strategy in NAFLD patients.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Dieta Alta en Grasa , Humanos , Inflamación , Hígado , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Pérdida de PesoRESUMEN
Digital PCR provides high sensitivity and unprecedented accuracy in DNA quantification, but current approaches require dedicated instrumentation and have limited opportunities for multiplexing. Here, we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy. Circular DNA strands, which may result from a wide range of molecular detection reactions, are captured on streptavidin-coated surfaces via hybridized biotinylated primers, followed by rolling circle amplification (RCA). The addition of 15% polyethylene glycol 4000 during RCA resulted in uniform, easily recorded reaction products. Immobilized DNA circles were visualized as RCA products with 100% efficiency, as determined by droplet digital PCR. We confirmed previous reports about the influence on RCA by sequence composition and size of RCA templates, and we developed an efficient one-step restaining procedure for sequential multiplexing using toehold-triggered DNA strand displacement. Finally, we exemplify applications of this digital readout platform by demonstrating more than three orders of magnitude improved sensitivity by digital measurement of prostate specific antigen (PSA) (detection threshold â¼100 pg/l), compared to a commercial enzyme-linked immunosorbent assay (ELISA) with analogue readout (detection threshold â¼500 ng/l), using the same antibody pair.
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ADN Circular/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Calicreínas/sangre , Técnicas de Amplificación de Ácido Nucleico/métodos , Antígeno Prostático Específico/sangre , Femenino , Humanos , Masculino , Hibridación de Ácido Nucleico/métodos , Estreptavidina/químicaRESUMEN
BACKGROUND: The lack of specificity and high degree of false positive and false negative rates when using mammographic screening for detecting early-stage breast cancer is a critical issue. Blood-based molecular assays that could be used in adjunct with mammography for increased specificity and sensitivity could have profound clinical impact. Our objective was to discover and independently verify a panel of candidate blood-based biomarkers that could identify the earliest stages of breast cancer and complement current mammographic screening approaches. METHODS: We used affinity hydrogel nanoparticles coupled with LC-MS/MS analysis to enrich and analyze low-abundance proteins in serum samples from 20 patients with invasive ductal carcinoma (IDC) breast cancer and 20 female control individuals with positive mammograms and benign pathology at biopsy. We compared these results to those obtained from five cohorts of individuals diagnosed with cancer in organs other than breast (ovarian, lung, prostate, and colon cancer, as well as melanoma) to establish IDC-specific protein signatures. Twenty-four IDC candidate biomarkers were then verified by multiple reaction monitoring (LC-MRM) in an independent validation cohort of 60 serum samples specifically including earliest-stage breast cancer and benign controls (19 early-stage (T1a) IDC and 41 controls). RESULTS: In our discovery set, 56 proteins were increased in the serum samples from IDC patients, and 32 of these proteins were specific to IDC. Verification of a subset of these proteins in an independent cohort of early-stage T1a breast cancer yielded a panel of 4 proteins, ITGA2B (integrin subunit alpha IIb), FLNA (Filamin A), RAP1A (Ras-associated protein-1A), and TLN-1 (Talin-1), which classified breast cancer patients with 100% sensitivity and 85% specificity (AUC of 0.93). CONCLUSIONS: Using a nanoparticle-based protein enrichment technology, we identified and verified a highly specific and sensitive protein signature indicative of early-stage breast cancer with no false positives when assessing benign and inflammatory controls. These markers have been previously reported in cell-ECM interaction and tumor microenvironment biology. Further studies with larger cohorts are needed to evaluate whether this biomarker panel improves the positive predictive value of mammography for breast cancer detection.
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Biomarcadores de Tumor/sangre , Neoplasias de la Mama/diagnóstico , Carcinoma Ductal de Mama/diagnóstico , Detección Precoz del Cáncer/métodos , Proteínas de la Matriz Extracelular/sangre , Adulto , Anciano , Biopsia , Mama/diagnóstico por imagen , Mama/patología , Neoplasias de la Mama/sangre , Carcinoma Ductal de Mama/sangre , Carcinoma Ductal de Mama/patología , Estudios de Casos y Controles , Estudios de Cohortes , Proteínas de la Matriz Extracelular/química , Femenino , Humanos , Masculino , Mamografía , Persona de Mediana Edad , Nanopartículas/química , Proteómica/métodosRESUMEN
Host proteins incorporated into virus particles have been reported to contribute to infectivity and tissue-tropism. This incorporation of host proteins is expected to be variable among viral particles, however, protein analysis at single-virus levels has been challenging. We have developed a method to detect host proteins incorporated on the surface of virions using the in situ proximity ligation assay (isPLA) with rolling circle amplification (RCA), employing oligonucleotide-conjugated antibody pairs. The technique allows highly selective and sensitive antibody-based detection of viral and host proteins on the surface of individual virions. We detected recombinant noninfectious sub-viral particles (SVPs) of tick-borne encephalitis virus (TBEV) immobilized in microtiter wells as fluorescent particles detected by regular fluorescence microscopy. Counting the particles in the images enabled us to estimate individual TBEV-SVP counts in different samples. Using isPLA we detected individual calnexin-, CD9-, CD81-, CD29- and CD59-positive SVPs among the viral particles. Our data suggests that a diversity of host proteins may be incorporated into TEBV, illustrating that isPLA with digital counting enables single-virus analysis of host protein incorporation.
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Virus de la Encefalitis Transmitidos por Garrapatas/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Proteínas/metabolismo , Línea Celular , Humanos , Proteínas/ultraestructura , Virión/metabolismo , Virión/ultraestructuraRESUMEN
The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity-free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.
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Inmunoensayo/métodos , Biotinilación , Humanos , Espectrometría de Masas , Persona de Mediana Edad , Plasma/química , Proteoma/análisis , Proteómica/métodosRESUMEN
The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (≤60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINA5, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.
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Fragmentos de Péptidos/análisis , Proteómica/métodos , Proteínas Recombinantes/análisis , Proteínas Sanguíneas/análisis , Cromatografía Liquida/métodos , Humanos , Marcaje Isotópico/métodos , Espectrometría de Masas en Tándem/métodos , Tripsina/metabolismoRESUMEN
BACKGROUND: Mammographic breast density is one of the strongest risk factors for breast cancer, but molecular understanding of how breast density relates to cancer risk is less complete. Studies of proteins in blood plasma, possibly associated with mammographic density, are well-suited as these allow large-scale analyses and might shed light on the association between breast cancer and breast density. METHODS: Plasma samples from 1329 women in the Swedish KARMA project, without prior history of breast cancer, were profiled with antibody suspension bead array (SBA) assays. Two sample sets comprising 729 and 600 women were screened by two different SBAs targeting a total number of 357 proteins. Protein targets were selected through searching the literature, for either being related to breast cancer or for being linked to the extracellular matrix. Association between proteins and absolute area-based breast density (AD) was assessed by quantile regression, adjusting for age and body mass index (BMI). RESULTS: Plasma profiling revealed linear association between 20 proteins and AD, concordant in the two sets of samples (p < 0.05). Plasma levels of seven proteins were positively associated and 13 proteins negatively associated with AD. For eleven of these proteins evidence for gene expression in breast tissue existed. Among these, ABCC11, TNFRSF10D, F11R and ERRF were positively associated with AD, and SHC1, CFLAR, ACOX2, ITGB6, RASSF1, FANCD2 and IRX5 were negatively associated with AD. CONCLUSIONS: Screening proteins in plasma indicates associations between breast density and processes of tissue homeostasis, DNA repair, cancer development and/or progression in breast cancer. Further validation and follow-up studies of the shortlisted protein candidates in independent cohorts will be needed to infer their role in breast density and its progression in premenopausal and postmenopausal women.
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Proteínas Sanguíneas/genética , Densidad de la Mama/genética , Neoplasias de la Mama/sangre , Proteómica , Adolescente , Adulto , Anciano , Índice de Masa Corporal , Mama/diagnóstico por imagen , Mama/patología , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Femenino , Humanos , Mamografía/métodos , Persona de Mediana Edad , Posmenopausia , Premenopausia , Factores de Riesgo , Adulto JovenRESUMEN
There is a clear clinical need for high-specificity plasma biomarkers for predicting risk of venous thromboembolism (VTE), but thus far, such markers have remained elusive. Utilizing affinity reagents from the Human Protein Atlas project and multiplexed immuoassays, we extensively analyzed plasma samples from 2 individual studies to identify candidate protein markers associated with VTE risk. We screened plasma samples from 88 VTE cases and 85 matched controls, collected as part of the Swedish "Venous Thromboembolism Biomarker Study," using suspension bead arrays composed of 755 antibodies targeting 408 candidate proteins. We identified significant associations between VTE occurrence and plasma levels of human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1), von Willebrand factor (VWF), glutathione peroxidase 3 (GPX3), and platelet-derived growth factor ß (PDGFB). For replication, we profiled plasma samples of 580 cases and 589 controls from the French FARIVE study. These results confirmed the association of VWF and PDGFB with VTE after correction for multiple testing, whereas only weak trends were observed for HIVEP1 and GPX3. Although plasma levels of VWF and PDGFB correlated modestly (ρ â¼ 0.30) with each other, they were independently associated with VTE risk in a joint model in FARIVE (VWF P < .001; PDGFB P = .002). PDGFΒ was verified as the target of the capture antibody by immunocapture mass spectrometry and sandwich enzyme-linked immunosorbent assay. In conclusion, we demonstrate that high-throughput affinity plasma proteomic profiling is a valuable research strategy to identify potential candidate biomarkers for thrombosis-related disorders, and our study suggests a novel association of PDGFB plasma levels with VTE.
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Proteómica , Proteínas Proto-Oncogénicas c-sis/sangre , Tromboembolia Venosa/sangre , Biomarcadores/sangre , Proteínas de Unión al ADN/sangre , Femenino , Glutatión Peroxidasa/sangre , Humanos , Masculino , Factores de Riesgo , Factores de Transcripción/sangre , Factor de von Willebrand/metabolismoRESUMEN
Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors for which identification and validation of biological markers may improve clinical management. The fraction of low-molecular-weight (LMW) circulating proteins and fragments of proteins is a rich source of new potential biomarkers. To identify circulating biomarkers useful for STS early diagnosis and prognosis, we analyzed 53 high-grade STS sera using hydrogel core-shell nanoparticles that selectively entrap LMW proteins by size exclusion and affinity chromatography, protect them from degradation and amplify their concentration for mass spectrometry detection. Twenty-two analytes mostly involved in inflammatory and immunological response, showed a progressive increase from benign to malignant STS with a relative difference in abundance, more than 50% when compared to healthy control. 16 of these were higher in metastatic compared to non-metastatic tumors. Cox's regression analysis revealed a statistical significant association between the abundance of lactotransferrin (LTF) and complement factor H-related 5 (CFHR5) and risk of metastasis. In particular, CFHR5 was associated with the risk of metastasis. The role of circulating proteins involved in metastatic progression will be crucial for a better understanding of STS biology and patient management.
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Proteínas Sanguíneas/análisis , Sarcoma/sangre , Sarcoma/diagnóstico , Biomarcadores de Tumor/sangre , Proteínas del Sistema Complemento/análisis , Diagnóstico Precoz , Humanos , Lactoferrina/análisis , Lactoferrina/sangre , Nanopartículas/química , Metástasis de la Neoplasia/diagnóstico , Pronóstico , Espectrometría de Masas en Tándem/métodosRESUMEN
Aiming at clinical studies of human diseases, antibody-assisted assays have been applied to biomarker discovery and toward a streamlined translation from patient profiling to assays supporting personalized treatments. In recent years, integrated strategies to couple and combine antibodies with mass spectrometry-based proteomic efforts have emerged, allowing for novel possibilities in basic and clinical research. Described in this review are some of the field's current and emerging immunocapture approaches from an affinity proteomics perspective. Discussed are some of their advantages, pitfalls and opportunities for the next phase in clinical and translational proteomics.
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Anticuerpos/química , Proteoma/aislamiento & purificación , Animales , Humanos , Inmunoprecipitación , Límite de Detección , Espectrometría de Masas , Unión Proteica , Proteómica/métodos , Investigación Biomédica TraslacionalRESUMEN
The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.
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Encéfalo/metabolismo , Líquido Cefalorraquídeo/metabolismo , Esclerosis Múltiple/sangre , Esclerosis Múltiple/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Anticuerpos , Técnica del Anticuerpo Fluorescente , Humanos , Países BajosRESUMEN
Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) Ë14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.
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Proteínas de la Membrana , SARS-CoV-2 , Humanos , Epítopos , Glicoproteínas de Membrana/metabolismo , Receptores Virales , ViriónRESUMEN
BACKGROUND: Self-sampling of dried blood spots (DBS) offers new routes to gather valuable health-related information from the general population. Yet, the utility of using deep proteome profiling from home-sampled DBS to obtain clinically relevant insights about SARS-CoV-2 infections remains largely unexplored. METHODS: Our study involved 228 individuals from the general Swedish population who used a volumetric DBS sampling device and completed questionnaires at home during spring 2020 and summer 2021. Using multi-analyte COVID-19 serology, we stratified the donors by their response phenotypes, divided them into three study sets, and analyzed 276 proteins by proximity extension assays (PEA). After normalizing the data to account for variances in layman-collected samples, we investigated the association of DBS proteomes with serology and self-reported information. RESULTS: Our three studies display highly consistent variance of protein levels and share associations of proteins with sex (e.g., MMP3) and age (e.g., GDF-15). Studying seropositive (IgG+) and seronegative (IgG-) donors from the first pandemic wave reveals a network of proteins reflecting immunity, inflammation, coagulation, and stress response. A comparison of the early-infection phase (IgM+IgG-) with the post-infection phase (IgM-IgG+) indicates several proteins from the respiratory system. In DBS from the later pandemic wave, we find that levels of a virus receptor on B-cells differ between seropositive (IgG+) and seronegative (IgG-) donors. CONCLUSIONS: Proteome analysis of volumetric self-sampled DBS facilitates precise analysis of clinically relevant proteins, including those secreted into the circulation or found on blood cells, augmenting previous COVID-19 reports with clinical blood collections. Our population surveys support the usefulness of DBS, underscoring the role of timing the sample collection to complement clinical and precision health monitoring initiatives.
The COVID-19 pandemic has posed multiple challenges to healthcare systems. A significant gap that remains is a lack of understanding of the impact of SARS-CoV-2 on individuals who did not seek or require hospitalization. To address this, we distribute self-sampling devices to random citizens, aiming to analyze how blood protein levels are affected in people who have had COVID-19 but had no or mild symptoms. Conducting multiple molecular measurements in dried blood, our study confirms clinically known markers and their relationship to infection stages, even if the donors themselves collect the sample. Our work highlights the potential of combining self-sampling with laboratory methods to provide useful information on human health. This convenient patient-centric sampling approach may potentially be useful when studying other diseases.
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The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.
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Anticuerpos Inmovilizados/química , Proteínas Sanguíneas/metabolismo , Proteínas de Fase Aguda , Especificidad de Anticuerpos , Quimiocinas CC/sangre , Bases de Datos de Proteínas , Humanos , Lipocalina 2 , Lipocalinas/sangre , Proteómica , Proteínas Proto-Oncogénicas/sangreRESUMEN
Pyruvate kinase (PK) is an important glycolytic enzyme that catalyzes the dephosphorylation of phosphoenolpyruvate to pyruvate. Human PK isozyme M2 (PKM2), a splice variant of M1, is overexpressed in many cancer cells, and PKM2 has been investigated as a potential tumor marker for diagnostic assays and as a target for cancer therapy. To facilitate identification and characterization of PK, we studied the enzyme from pancreatic cancer cells and normal pancreatic duct cells by electrophoresis and mass spectrometry, and identified multiple O-methylated residues from PK. These findings advance our knowledge of the biochemical properties of PK and will be important in understanding its biological function in cells.
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Biomarcadores de Tumor/análisis , Espectrometría de Masas/métodos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/enzimología , Piruvato Quinasa/análisis , Piruvato Quinasa/química , Humanos , Metilación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
Blood sampling is a common practice to monitor health, but it entails a series of drawbacks for patients including pain and discomfort. Thus, there is a demand for more convenient ways to obtain samples. Modern analytical techniques enable monitoring of multiple bioanalytes in smaller samples, opening possibilities for new matrices, and microsampling technologies to be adopted. Interstitial fluid (ISF) is an attractive alternative matrix that shows good correlation with plasma concentration dynamics for several analytes and can be sampled in a minimally invasive and painless manner from the skin at the point-of-care. However, there is currently a lack of sampling devices compatible with clinical translation. Here, to tackle state-of-the-art limitations, a cost-effective and compact single-microneedle-based device designed to painlessly collect precisely 1.1 µL of dermal ISF within minutes is presented. The fluid is volume-metered, dried, and stably stored into analytical-grade paper within the microfluidic device. The obtained sample can be mailed to a laboratory, quantitatively analyzed, and provide molecular insights comparable to blood testing. In a human study, the possibility to monitor various classes of molecular analytes is demonstrated in ISF microsamples, including caffeine, hundreds of proteins, and SARS-CoV-2 antibodies, some being detected in ISF for the first time.