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Fibroblast growth factors (FGFs) are paracrine or endocrine signaling proteins that, activated by their ligands, elicit a wide range of health and disease-related processes, such as cell proliferation and the epithelial-to-mesenchymal transition. The detailed molecular pathway dynamics that coordinate these responses have remained to be determined. To elucidate these, we stimulated MCF-7 breast cancer cells with either FGF2, FGF3, FGF4, FGF10, or FGF19. Following activation of the receptor, we quantified the kinase activity dynamics of 44 kinases using a targeted mass spectrometry assay. Our system-wide kinase activity data, supplemented with (phospho)proteomics data, reveal ligand-dependent distinct pathway dynamics, elucidate the involvement of not earlier reported kinases such as MARK, and revise some of the pathway effects on biological outcomes. In addition, logic-based dynamic modeling of the kinome dynamics further verifies the biological goodness-of-fit of the predicted models and reveals BRAF-driven activation upon FGF2 treatment and ARAF-driven activation upon FGF4 treatment.
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Fator 2 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fosforilação , Proliferação de Células , Espectrometria de MassasRESUMO
BACKGROUND: Differentiating between a normal intrauterine pregnancy (IUP) and abnormal conditions including early pregnancy loss (EPL) or ectopic pregnancy (EP) is a major clinical challenge in early pregnancy. Currently, serial ß-human chorionic gonadotropin (ß-hCG) and progesterone are the most commonly used plasma biomarkers for evaluating pregnancy prognosis when ultrasound is inconclusive. However, neither biomarker can predict an EP with sufficient and reproducible accuracy. Hence, identification of new plasma biomarkers that can accurately diagnose EP would have great clinical value. METHODS: Plasma was collected from a discovery cohort of 48 consenting women having an IUP, EPL, or EP. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by a label-free proteomics analysis to identify significant changes between pregnancy outcomes. A panel of 14 candidate biomarkers were then verified in an independent cohort of 74 women using absolute quantitation by targeted parallel reaction monitoring mass spectrometry (PRM-MS) which provided the capacity to distinguish between closely related protein isoforms. Logistic regression and Lasso feature selection were used to evaluate the performance of individual biomarkers and panels of multiple biomarkers to predict EP. RESULTS: A total of 1391 proteins were identified in an unbiased plasma proteome discovery. A number of significant changes (FDR ≤ 5%) were identified when comparing EP vs. non-EP (IUP + EPL). Next, 14 candidate biomarkers (ADAM12, CGA, CGB, ISM2, NOTUM, PAEP, PAPPA, PSG1, PSG2, PSG3, PSG9, PSG11, PSG6/9, and PSG8/1) were verified as being significantly different between EP and non-EP in an independent cohort (FDR ≤ 5%). Using logistic regression models, a risk score for EP was calculated for each subject, and four multiple biomarker logistic models were identified that performed similarly and had higher AUCs than models with single predictors. CONCLUSIONS: Overall, four multivariable logistic models were identified that had significantly better prediction of having EP than those logistic models with single biomarkers. Model 4 (NOTUM, PAEP, PAPPA, ADAM12) had the highest AUC (0.987) and accuracy (96%). However, because the models are statistically similar, all markers in the four models and other highly correlated markers should be considered in further validation studies.
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Dietary supplements are legally considered foods despite frequently including medicinal plants as ingredients. Currently, the consumption of herbal dietary supplements, also known as plant food supplements (PFS), is increasing worldwide and some raw botanicals, highly demanded due to their popularity, extensive use, and/or well-established pharmacological effects, have been attaining high prices in the international markets. Therefore, botanical adulteration for profit increase can occur along the whole PFS industry chain, from raw botanicals to plant extracts, until final PFS. Besides the substitution of high-value species, unintentional mislabeling can happen in morphologically similar species. Both cases represent a health risk for consumers, prompting the development of numerous works to access botanical adulterations in PFS. Among different approaches proposed for this purpose, mass spectrometry (MS)-based techniques have often been reported as the most promising, particularly when hyphenated with chromatographic techniques. Thus, this review aims at describing an overview of the developments in this field, focusing on the applications of MS-based techniques to targeted and untargeted analysis to detect botanical adulterations in plant materials, extracts, and PFS.
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Suplementos Nutricionais , Plantas Medicinais , Espectrometria de Massas/métodos , Contaminação de MedicamentosRESUMO
Plant polyphenols have beneficial antioxidant effects on human health; practices aimed at preserving their content in foods and/or reusing food by-products are encouraged. The impact of the traditional practice of the water curing procedure of chestnuts, which prevents insect/mould damage during storage, was studied to assess the release of polyphenols from the fruit. Metabolites extracted from pericarp and integument tissues or released in the medium from the water curing process were analyzed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray-quadrupole-time of flight-mass spectrometry (ESI-qTOF-MS). This identified: (i) condensed and hydrolyzable tannins made of (epi)catechin (procyanidins) and acid ellagic units in pericarp tissues; (ii) polyphenols made of gallocatechin and catechin units condensed with gallate (prodelphinidins) in integument counterparts; (iii) metabolites resembling those reported above in the wastewater from the chestnut curing process. Comparative experiments were also performed on aqueous media recovered from fruits treated with processes involving: (i) tap water; (ii) tap water containing an antifungal Lb. pentosus strain; (iii) wastewater from a previous curing treatment. These analyses indicated that the former treatment determines a 6-7-fold higher release of polyphenols in the curing water with respect to the other ones. This event has a negative impact on the luster of treated fruits but qualifies the corresponding wastes as a source of antioxidants. Such a phenomenon does not occur in wastewater from the other curing processes, where the release of polyphenols was reduced, thus preserving the chestnut's appearance. Polyphenol profiling measurements demonstrated that bacterial presence in water hampered the release of pericarp metabolites. This study provides a rationale to traditional processing practices on fruit appearance and qualifies the corresponding wastes as a source of bioactive compounds for other nutraceutical applications.
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Aesculus/química , Antioxidantes/química , Extratos Vegetais/química , Polifenóis/química , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Biflavonoides/química , Biflavonoides/isolamento & purificação , Catequina/química , Catequina/isolamento & purificação , Frutas/química , Humanos , Nozes/química , Extratos Vegetais/farmacologia , Polifenóis/isolamento & purificação , Polifenóis/metabolismo , Proantocianidinas/química , Proantocianidinas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Taninos/química , Água/químicaRESUMO
Diacyl glycerophospholipids (GPs) belong to the most abundant lipid species in living organisms and consist of a glycerol backbone with fatty acyl groups in sn-1 and sn-2 and a polar head group in the sn-3 position. Regioisomeric mixed diacyl GPs have the same fatty acyl composition but differ in their allocation to sn-1 or sn-2 of the glycerol unit. In-depth analysis of regioisomeric mixed diacyl GP species composed of fatty acyl moieties that are similar in length and degree of saturation typically requires either chemical derivatization or sophisticated analytical instrumentation, since these types of regioisomers are not well resolved under standard ultra-performance liquid chromatography (UPLC) conditions. Here, we introduce a simple and fast method for diacyl GP regioisomer analysis employing UPLC tandem mass spectrometry (MS/MS). This GP regioisomer analysis is based both on minor chromatographic retention time shifts and on major differences in relative abundances of the two fatty acyl anion fragments observed in MS/MS. To monitor these differences with optimal precision, MS/MS spectra are recorded continuously over the UPLC elution profile of the lipid species of interest. Quantification of relative abundances of the regioisomers was performed by algorithms that we have developed for this purpose. The method was applied to commercially available mixed diacyl GP standards and to total lipid extracts of Escherichia coli (E. coli) and bovine liver. To validate our results, we determined regioisomeric ratios of phosphatidylcholine (PC) standards using phospholipase A2-specific release of fatty acids from the sn-2 position of the glycerol backbone. Our results show that most analyzed mixed diacyl GPs of biological origin exhibit significantly higher regioisomeric purity than synthetic lipid standards. In summary, this method can be implemented in routine LC-MS/MS-based lipidomics workflows without the necessity for additional chemical additives, derivatizations, or instrumentation.
Assuntos
Cromatografia Líquida/métodos , Glicerofosfolipídeos/análise , Glicerofosfolipídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Escherichia coli/química , Glicerofosfolipídeos/normas , Fígado/química , Padrões de Referência , EstereoisomerismoRESUMO
AIMS/HYPOTHESIS: Gestational diabetes mellitus (GDM) is associated with an increased risk of pre-eclampsia, macrosomia and the future development of type 2 diabetes mellitus in both mother and child. Although an early and accurate prediction of GDM is needed to allow intervention and improve perinatal outcome, no single protein biomarker has yet proven useful for this purpose. In the present study, we hypothesised that multimarker panels of serum proteins can improve first-trimester prediction of GDM among obese and non-obese women compared with single markers. METHODS: A nested case-control study was performed on first-trimester serum samples from 199 GDM cases and 208 controls, each divided into an obese group (BMI ≥27 kg/m(2)) and a non-obese group (BMI <27 kg/m(2)). Based on their biological relevance to GDM or type 2 diabetes mellitus or on their previously reported potential as biomarkers for these diseases, a number of proteins were selected for targeted nano-flow liquid chromatography (LC) MS analysis. This resulted in the development and validation of a 25-plex multiple reaction monitoring (MRM) MS assay. RESULTS: After false discovery rate correction, six proteins remained significantly different (p<0.05) between obese GDM patients (n=135) and BMI-matched controls (n=139). These included adiponectin, apolipoprotein M and apolipoprotein D. Multimarker models combining protein levels and clinical data were then constructed and evaluated by receiver operating characteristic (ROC) analysis. For the obese, non-obese and all GDM groups, these models achieved marginally higher AUCs compared with adiponectin alone. CONCLUSIONS/INTERPRETATION: Multimarker models combining protein markers and clinical data have the potential to predict women at a high risk of developing GDM.
Assuntos
Diabetes Gestacional/sangue , Diabetes Gestacional/diagnóstico , Adipocinas/sangue , Apolipoproteínas/sangue , Estudos de Casos e Controles , Cromatografia Líquida , Feminino , Humanos , Obesidade/sangue , Gravidez , Primeiro Trimestre da Gravidez/sangueRESUMO
Experimental determination of absolute protein amounts is becoming increasingly important for the establishment and validation of biomarkers, and systems biology approaches aimed at a quantitative description of a biological process. Residing at compartmental or cellular barriers, and acting as prominent drug targets, integral membranes proteins, being completely embedded in the lipid bilayer, possess characteristic physicochemical properties and are often in low abundance. These features challenge the quantification with targeted MS and the ability to accurately determine the amount of membrane proteins with high sensitivity. This review summarizes the current status of targeted membrane protein quantification with emphasis on sample preparation beforehand MS. From the beginning to the end of a usual sample preparation workflow, consisting essentially of reference point selection, cell lysis, digestion, and addition of suitable isotope-labeled standards, general and particular challenges for membrane proteins will be discussed step by step. Based on the presentation of current achievements, possible measures to better address these challenges and future avenues of targeted membrane proteomics are presented.
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Marcação por Isótopo , Espectrometria de Massas , Proteínas de Membrana/análise , ProteômicaRESUMO
Citri Reticulatae Pericarpium is a traditional Chinese medicine with extremely high health benefits as well as clinical value. In vivo and in vitro tests have proved that its main active secondary metabolites are flavonoids. However, they have not been comprehensively analyzed up to now mainly due to lack of suitable analysis method. To solve this problem, a novel strategy based on precursor ions locked and targeted MS/MS analysis was proposed. Firstly, the database of the flavonoids previously isolated from Citri Reticulatae Pericarpium was established to obtain the characteristics of their precursor ions. Secondly, after performing the full MS scan of the extract, all compounds in the total ion chromatogram were extracted by Compound Discoverer software. Thirdly, the precursor ions of the flavonoids were locked from the extracted compounds according to their characteristics, forming a precursor ions list. Finally, the precursor ions in the constructed list were performed targeted MS/MS analysis for structures characterization. As a result, total 187 flavonoids were successfully identified, and except for flavones, flavonols as well as dihydroflavones, some chalcones were also characterized from Citri Reticulatae Pericarpium for the first time.
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Citrus , Flavonoides , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Flavonoides/análise , Flavonoides/química , Citrus/química , Íons , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/análiseRESUMO
We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms.
Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Humanos , Espectrometria de Massas/métodos , Ligação Proteica , Espectrometria de Massas em Tandem/métodosRESUMO
To comprehensively study the ginsenosides distribution in the various tissues of American ginseng, the qualitative and quantitative-targeted and nontargeted mass spectroscopic methods were established using the high-performance liquid chromatography coupled with Qtrap triple quadrupole mass spectrometry (HPLC-QtrapQQQ-MS). The total ginsenosides of the root, stem, and leaf of American ginseng were determined by a colorimetric method, and the contents showed the order from high to low root, stem, and leaf. Eighty-two kinds of ginsenosides were detected in the different parts of American ginseng by enhanced mass scan-information-dependent data acquisition (IDA)-enhanced product ion (EPI) scan mode, including 69 from the root, 62 from the stem, and 48 from the leaf. An HPLC-multiple reaction monitoring (MRM) method was established, and 28 representative ginsenosides were further quantified in the three parts. Nearly all ginsenosides had the highest contents in the root and the lowest content in the leaf. Three types of ginsenosides (protopanaxadiol [PPD]-, protopanaxatiol [PPT]-, and oleanolic acid [OA]-types) were analyzed by precursor ion-IDA-EPI and MRM-IDA-EPI scan modes. Root had the most abundant ginsenosides in PPD- and PPT-type ginsenosides. Meanwhile, the OA-type ginsenosides are significantly enriched in the stem and leaf of American ginseng. The results provided a supplement to the quality assessment of American ginseng. PRACTICAL APPLICATION: The distribution profile of ginsenosides in the parts of American ginseng is different. Except for the root, the stem, and leaf of American ginseng have the most abundant ginsenosides in oleanolic acid type. The results reported herein can help the manufacturers choose appropriate materials to extract the ginsenosides.
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Ginsenosídeos , Ácido Oleanólico , Panax , Espectrometria de Massas em Tandem/métodos , Panax/química , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Oxidized cholesteryl ester (OxCE) is produced by the oxidation of cholesteryl ester (CE) in the cores of lipoproteins. OxCE production and oxidative stress have been largely associated with breast cancer. Herein, we developed a novel reverse-phase liquid chromatography coupling quadrupole time-of-flight mass spectrometry (RPLCâQ-TOFâMS) method based on the iterative acquisition mode and used the MS/MS mode for deep mining and simultaneous quantification of cholesterol (Chol), CEs and OxCEs in human serum. A mathematical model was used to globally profile 57 molecular species of both CEs and OxCEs in the serum of both healthy volunteers and patients with breast cancer, and the qualitative results were verified based on the retention regularity. An abnormal elevation of OxCEs was found in serum samples of breast cancer patients, where OxCEs were produced by the oxidation of the fatty acyl chain of CE (20:4), such as CE (20:1)+3O, CE (20:2)+2O and CE (20:3)+O, which could be regarded as biomarkers. This comprehensive method for the global profiling of Chol, OxCEs and CEs sheds light on the role OxCEs and CEs play in breast cancer and has enabled the discovery of breast cancer biomarkers.
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Neoplasias da Mama , Ésteres do Colesterol , Biomarcadores Tumorais , Cromatografia Líquida , Feminino , Humanos , Espectrometria de Massas em TandemRESUMO
The presence and abundance of viral proteins within host cells are part of the essential signatures of the cellular stages of viral infections. However, methods that can comprehensively detect and quantify these proteins are still limited, particularly for viruses with large protein coding capacity. Here, we design and experimentally validate a mass spectrometry-based Targeted herpesviRUS proTEin Detection (TRUSTED) assay for monitoring human viruses representing the three Herpesviridae subfamilies-herpes simplex virus type 1, human cytomegalovirus (HCMV), and Kaposi sarcoma-associated herpesvirus. We demonstrate assay applicability for (1) capturing the temporal cascades of viral replication, (2) detecting proteins throughout a range of virus concentrations and in in vivo models of infection, (3) assessing the effects of clinical therapeutic agents and sirtuin-modulating compounds, (4) studies using different laboratory and clinical viral strains, and (5) discovering a role for carbamoyl phosphate synthetase 1 in supporting HCMV replication.
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Herpesvirus Humano 1 , Herpesvirus Humano 8 , Citomegalovirus , Humanos , Espectrometria de Massas , Replicação ViralRESUMO
Increasing number of studies report the relevance of protein Ser/Thr/Tyr phosphorylation in bacterial physiology, yet the analysis of this type of modification in bacteria still presents a considerable challenge. Unlike in eukaryotes, where tens of thousands of phosphorylation events likely occupy more than two thirds of the proteome, the abundance of protein phosphorylation is much lower in bacteria. Even the state-of-the-art phosphopeptide enrichment protocols fail to remove the high background of abundant unmodified peptides, leading to low signal intensity and undersampling of phosphopeptide precursor ions in consecutive data-dependent MS runs. Consequently, large-scale bacterial phosphoproteomic datasets often suffer from poor reproducibility and a high number of missing values. Here we explore the application of parallel reaction monitoring (PRM) on a Q Exactive mass spectrometer in bacterial phosphoproteome analysis, focusing especially on run-to-run sampling reproducibility. In multiple measurements of identical phosphopeptide-enriched samples, we show that PRM outperforms data-dependent acquisition (DDA) in terms of detection frequency, reaching almost complete sampling efficiency, compared to 20% in DDA. We observe a similar trend over multiple heterogeneous phosphopeptide-enriched samples and conclude that PRM shows a great promise in bacterial phosphoproteomics analyses where reproducible detection and quantification of a relatively small set of phosphopeptides is desired. SIGNIFICANCE: Bacterial phosphorylated peptides occur in low abundance compared to their unmodified counterparts, and are therefore rarely reproducibly detected in shotgun (DDA) proteomics measurements. Here we show that parallel reaction monitoring complements DDA analyses and makes detection of known, targeted phosphopeptides more reproducible. This will be of significance in replicated MS measurements that have a goal to reproducibly detect and quantify phosphopeptides of interest.
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Proteínas de Bactérias/análise , Fosfopeptídeos/análise , Proteoma/análise , Proteômica/métodos , Proteínas de Bactérias/química , Escherichia coli/química , Escherichia coli/metabolismo , Estudos de Avaliação como Assunto , Fosfopeptídeos/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Fosforilação , Proteômica/instrumentação , Reprodutibilidade dos TestesRESUMO
Anabolic and androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their time of action. The authors' laboratory has previously reported an UHPLC/HRMS method using quadrupole-Orbitrap mass spectrometer in full scan and parallel reaction monitoring (PRM) mode for the detection of 48 AAS and/or their esters in horse hair. However, two injections were required due to the long duty cycle time. In this paper, an UHPLC/HRMS method using multiplexed targeted MS2 mode was developed and validated to improve the coverage to 65 AAS and/or their esters in a single injection. In addition, a GC/MS/MS method in selected reaction monitoring (SRM) mode was developed to screen for another seven AAS and/or their esters not adequately covered by the UHPLC/HRMS method using the same sample extract after derivatisation with pentafluoropropionic anhydride. The UHPLC/HRMS and GC/MS/MS methods in combination allowed the detection of 72 AAS and/or their esters with estimated limits of detection down to sub to low ppb levels with good interday precision. Method applicability was demonstrated by the detection of boldione and 4-androstenedione in two out-of-competition hair samples and testosterone propionate in a referee hair sample.
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Cromatografia Líquida de Alta Pressão , Ésteres/análise , Cromatografia Gasosa-Espectrometria de Massas , Cabelo/química , Esteroides/análise , Espectrometria de Massas em Tandem , Androstenodiona/análise , Animais , Dopagem Esportivo , Ésteres/química , Cavalos , Esteroides/química , Propionato de Testosterona/análiseRESUMO
Following the rapid expansion of the proteomics field, the investigation of post translational modifications (PTM) has become extremely popular changing our perspective of how proteins constantly fine tune cellular functions. Reversible protein phosphorylation plays a pivotal role in virtually all biological processes in the cell and it is one the most characterized PTM up to date. During the last decade, the development of phosphoprotein/phosphopeptide enrichment strategies and mass spectrometry (MS) technology has revolutionized the field of phosphoproteomics discovering thousands of new site-specific phosphorylations and unveiling unprecedented evidence about their modulation under distinct cellular conditions. The field has expanded so rapidly that the use of traditional methods to validate and characterize the biological role of the phosphosites is not feasible any longer. Targeted MS holds great promise for becoming the method of choice to study with high precision and sensitivity already known site-specific phosphorylation events. This review summarizes the contribution of large-scale unbiased MS analyses and highlights the need of targeted MS-based approaches for follow-up investigation. Additionally, the article illustrates the biological relevance of protein phosphorylation by providing examples of disease-related phosphorylation events and emphasizes the benefits of applying targeted MS in clinics for disease diagnosis, prognosis and drug-response evaluation.
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Espectrometria de Massas/métodos , Fosfoproteínas/análise , Proteômica/métodos , Sequência de Aminoácidos , Animais , Humanos , Fosfopeptídeos/análise , Fosforilação , Proteoma/análiseRESUMO
SCOPE: Anti-inflammatory effects of coffee consumption have been reported to be caused by caffeine and adenosine receptor signaling. However, contradictory effects have been observed. Many kinds of chronic diseases are linked to inflammation; therefore a profound understanding of potential effects of coffee consumption is desirable. METHODS AND RESULTS: We performed ex vivo experiments with eight individuals investigating peripheral blood mononuclear cells isolated from venous blood before and after coffee consumption, as well as in vitro experiments applying caffeine on isolated cells. After in vitro inflammatory stimulation of the cells, released cytokines, chemokines, and eicosanoids were determined and quantified using targeted mass spectrometric methods. Remarkably, the release of inflammation mediators IL6, IL8, GROA, CXCL2, CXCL5 as well as PGA2, PGD2, prostaglandin E2 (PGE2), LTC4, LTE4, and 15S-HETE was significantly affected after coffee consumption. While in several individuals coffee consumption or caffeine treatment caused significant downregulation of most inflammation mediators, in other healthy individuals exactly the opposite effects were observed. CONCLUSION: Ruling out age, sex, coffee consumption habits, the metabolic kinetics of caffeine in blood and the individual amount of regulatory T cells or CD39 expression as predictive parameters, we demonstrated here that coffee consumption may have significant pro- or anti-inflammatory effects in an individual fashion.
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Café/química , Inflamação/sangue , Adulto , Cafeína/administração & dosagem , Células Cultivadas , Quimiocinas/sangue , Quimiocinas/genética , Citocinas/sangue , Citocinas/genética , Eicosanoides/sangue , Eicosanoides/genética , Feminino , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: While HIV-1 TAT peptide-conjugation shows great promise on improving intracellular delivery of biotherapeutics in vitro and in vivo, quantification of TAT-fusion therapeutics in biological matrices represents a daunting challenge. MATERIALS & METHODS: A sensitive MS approach for accurate quantification of intact TAT-fusion protein/polypeptide in plasma was developed. i) A semi-automated 96-well ion-exchange solid phase extraction was developed; ii) a rapid LC separation on C4 was devised; iii) a TAT-fusion analog was constructed as internal standard. RESULTS: We reported that low percentage of supercharging reagents enabled a significant sensitivity improvement of MS for intact TAT-fusion protein/polypeptide analysis. We showed a proof of concept by successfully developing a sensitive LC/MRM-MS method for quantifying GAP161, a TAT-conjugating RasGAP mimics, in rat plasma. CONCLUSION: This work represents the first quantification of TAT-fusion therapeutics in biological samples by an LC-MS based method.