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1.
Anal Chim Acta ; 1178: 338551, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482862

RESUMO

Single-cell analysis can allow for an in-depth understanding of diseases, diagnostics, and aid the development of therapeutics. However, single-cell analysis is challenging, as samples are both extremely limited in size and complex. But the concept is gaining promise, much due to novel sample preparation approaches and the ever-improving field of mass spectrometry. The mass spectrometer's output is often linked to the preceding compound separation step, typically being liquid chromatography (LC). In this review, we focus on LC's role in single-cell omics. Particle-packed nano LC columns (typically 50-100 µm inner diameter) have traditionally been the tool of choice for limited samples, and are also used for single cells. Several commercial products and systems are emerging with single cells in mind, featuring particle-packed columns or miniaturized pillar array systems. In addition, columns with inner diameters as narrow as 2 µm are being explored to maximize sensitivity. Hence, LC column down-scaling is a key focus in single-cell analysis. But narrow columns are associated with considerable technical challenges, while single cell analysis may be expected to become a "routine" service, requiring higher degrees of robustness and throughput. These challenges and expectations will increase the need and attention for the development (and even the reinvention) of alternative nano LC column formats. Therefore, monolith columns and even open tubular columns may finally find their "killer-application" in single cell analysis.


Assuntos
Análise de Célula Única , Cromatografia Líquida , Espectrometria de Massas
2.
J Proteome Res ; 20(8): 4010-4021, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34296888

RESUMO

Dried blood spot (DBS) metabolite analysis is a central tool for the clinic, e.g., newborn screening. Instead of applying multiple analytical methods, a single liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolites spanning from highly polar glucose to hydrophobic long-chain acylcarnitines. For liquid chromatography, a diphenyl column and a multi-linear solvent gradient operated at elevated flow rates allowed for an even-spread resolution of diverse metabolites. Injecting moderate volumes of DBS organic extracts directly, in contrast to evaporation and reconstitution, provided substantial increases in analyte recovery. Q Exactive MS settings were also tailored for sensitivity increases, and the method allowed for analyte retention time and peak area repeatabilities of 0.1-0.4 and 2-10%, respectively, for a wide polarity range of metabolites (log P -4.4 to 8.8). The method's performance was suited for both untargeted analysis and targeted approaches evaluated in clinically relevant experiments.


Assuntos
Metaboloma , Metabolômica , Cromatografia Líquida , Teste em Amostras de Sangue Seco , Humanos , Recém-Nascido , Espectrometria de Massas
3.
Anal Chem ; 93(7): 3576-3585, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33534551

RESUMO

Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 µL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.


Assuntos
Organoides , Preparações Farmacêuticas , Fígado , Espectrometria de Massas , Membranas Artificiais
4.
Sci Rep ; 11(1): 273, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431985

RESUMO

The eye lens is a unique organ as no cells can be replaced throughout life. This makes it decisive that the lens is protected against damaging UV-radiation. An ultraviolet (UV)-absorbing compound of unknown identity is present in the aqueous humor of geese (wild and domestic) and other birds flying at high altitudes. A goose aqueous humor extract, that was believed to contain the UV protective compound which was designated as "compound X", was fractionated and examined using a variety of spectroscopic techniques including LC-MS and high field one- and two dimensional-NMR methods. A series of compounds were identified but none of them appeared to be the UV protective "compound X". It may be that the level of the UV protective compound in goose aqueous humor is much less than the compounds identified in our investigation, or it may have been degraded by the isolation and chromatographic purification protocols used in our investigations.


Assuntos
Aves , Olho/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Humor Aquoso/metabolismo , Ácido Ascórbico/metabolismo , Aves/metabolismo , Olho/metabolismo , Voo Animal
5.
Curr Diab Rep ; 20(12): 72, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33206261

RESUMO

PURPOSE OF REVIEW: Human pancreas-on-a-chip (PoC) technology is quickly advancing as a platform for complex in vitro modeling of islet physiology. This review summarizes the current progress and evaluates the possibility of using this technology for clinical islet transplantation. RECENT FINDINGS: PoC microfluidic platforms have mainly shown proof of principle for long-term culturing of islets to study islet function in a standardized format. Advancement in microfluidic design by using imaging-compatible biomaterials and biosensor technology might provide a novel future tool for predicting islet transplantation outcome. Progress in combining islets with other tissue types gives a possibility to study diabetic interventions in a minimal equivalent in vitro environment. Although the field of PoC is still in its infancy, considerable progress in the development of functional systems has brought the technology on the verge of a general applicable tool that may be used to study islet quality and to replace animal testing in the development of diabetes interventions.


Assuntos
Diabetes Mellitus Tipo 1 , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Humanos , Dispositivos Lab-On-A-Chip , Pâncreas , Tecnologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-32971370

RESUMO

3', 5' - Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1-500 ng/mL cell extract.


Assuntos
Cromatografia de Fase Reversa/métodos , AMP Cíclico/análise , AMP Cíclico/metabolismo , Técnicas Citológicas/métodos , Espectrometria de Massas em Tandem/métodos , Brefeldina A , Toxina da Cólera , Células HT29 , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
7.
Front Chem ; 7: 835, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31850321

RESUMO

Prior to mass spectrometry, on-line sample preparation can be beneficial to reduce manual steps, increase speed, and enable analysis of limited sample amounts. For example, bottom-up proteomics sample preparation and analysis can be accelerated by digesting proteins to peptides in an on-line enzyme reactor. We here focus on low-backpressure 100 µm inner diameter (ID) × 160 mm, 180 µm ID × 110 mm or 250 µm ID × 140 mm vinyl azlactone-co-ethylene dimethacrylate [poly(VDM-co-EDMA)] monoliths as supports for immobilizing of additional molecules (i.e., proteases or drugs), as the monolith was expected to have few unspecific interactions. For on-line protein digestion, monolith supports immobilized with trypsin enzyme were found to be suited, featuring the expected characteristics of the material, i.e., low backpressure and low carry-over. Serving as a functionalized sample loop, the monolith units were very simple to connect on-line with liquid chromatography. However, for on-line target deconvolution, the monolithic support immobilized with a Wnt pathway inhibitor was associated with numerous secondary interactions when exploring the possibility of selectively trapping target proteins by drug-target interactions. Our initial observations suggest that (poly(VDM-co-EDMA)) monoliths are promising for e.g., on-line bottom-up proteomics, but not a "fit-for-all" material. We also discuss issues related to the repeatability of monolith-preparations.

8.
Analyst ; 144(24): 7090-7104, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31728480

RESUMO

Nano liquid chromatography (nanoLC), with columns having an inner diameter (ID) of ≤100 µm, can provide enhanced sensitivity and enable analysis of limited samples. NanoLC has become an established tool in omics research, and is gaining ground in other applications as well. There are several variants and formats of nanoLC columns, including packed columns, monoliths, open tubular columns, and the pillar array format. Most applications are done with packed columns, while e.g. the monolith and open tubular columns are still less established as routine tools. The pillar array format is a new variant with excellent resolution and low backpressure, and has recently been commercialized and used for bio-applications. In this minireview, we summarize and discuss recent research on nanoLC column development and uses, focusing on literature between 2016 and medio 2019.

9.
Psychoneuroendocrinology ; 107: 225-231, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31163380

RESUMO

Since its discovery more than a century ago, oxytocin has become one of the most intensively studied molecules in behavioral biology. In the last five years, Psychoneuroendocrinology has published more than 500 articles with oxytocin in the title, with many of these articles including measures of endogenous oxytocin concentrations. Despite longstanding interest, methods of measuring endogenous oxytocin are still in active development. The widely varying oxytocin concentrations detected by different approaches to measurement - and lack of correlation among these techniques - has led to controversy and confusion. We identify features of oxytocin that may help to explain why various approaches may be differentially sensitive to diverse conformational states of the oxytocin molecule. We propose that discrepancies in data generated by different methods of measurement are not necessarily an indicator that some methods are valid whereas others are not. Rather, we propose that current challenges in the measurement of oxytocin may be analogous to the parable of the blind men and the elephant, with different methods of sample preparation and measurement being sensitive to different states in which the oxytocin molecule can exist.


Assuntos
Ocitocina/análise , Ocitocina/metabolismo , Ocitocina/fisiologia , Animais , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Imunoensaio/métodos , Reprodutibilidade dos Testes , Manejo de Espécimes/métodos , Vasopressinas/metabolismo , Vasopressinas/fisiologia
10.
J Sep Sci ; 42(11): 1960-1961, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31050192
11.
J Proteome Res ; 18(5): 2012-2020, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30964684

RESUMO

Glioblastoma is the most common and malignant brain tumor, and current therapies confer only modest survival benefits. A major obstacle is our ability to monitor treatment effect on tumors. Current imaging modalities are ambiguous, and repeated biopsies are not encouraged. To scout for markers of treatment response, we used NMR spectroscopy to study the effects of a survivin inhibitor on the metabolome of primary glioblastoma cancer stem cells. Applying high resolution NMR spectroscopy (1H resonance frequency: 800.03 MHz) to just 3 million cells per sample, we achieved sensitive and high resolving determinations of, e.g., amino acids, nucleosides, and constituents of the citric acid cycle. For control samples that were cultured, prepared, and measured at varying dates, peak area relative standard deviations were 15-20%. Analyses of unfractionated lysates were performed for straightforward compound identification with COLMAR and HMDB databases. Principal component analysis revealed that citrate levels were clearly upregulated in nonresponsive cells, while lactate levels substantially decreased following treatment for both responsive and nonresponsive cells. Hence, lactate and citrate may be potential markers of successful drug uptake and poor response to survivin inhibitors, respectively. Our metabolomics approach provided alternative biomarker candidates compared to spectrometry-based proteomics, underlining benefits of complementary methodologies. These initial findings make a foundation for exploring in vivo MR spectroscopy (MRS) of brain tumors, as citrate and lactate are MRS-visible. In sum, NMR metabolomics is a tool for addressing glioblastoma.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Ácido Cítrico/metabolismo , Glioblastoma/tratamento farmacológico , Imidazóis/uso terapêutico , Ácido Láctico/metabolismo , Metaboloma , Naftoquinonas/uso terapêutico , Biomarcadores Farmacológicos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Sobrevivência Celular/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Espectroscopia de Ressonância Magnética , Terapia de Alvo Molecular/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Cultura Primária de Células , Análise de Componente Principal , Survivina/antagonistas & inibidores , Survivina/genética , Survivina/metabolismo
12.
Ther Drug Monit ; 41(4): 519-527, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30807539

RESUMO

BACKGROUND: The opioid analgesic fentanyl and its analogues pose a major health concern due to its high potency and the increasing number of overdose deaths worldwide. The analogues of fentanyl may differ in potency, toxicity, and legal status, and it is therefore important to develop analytical methods for their correct identification. This can be challenging since many fentanyl analogues are structural isomers. Two fentanyl isomers that have been in the spotlight lately due to difficulties regarding separation and identification are cyclopropylfentanyl and crotonylfentanyl, which have been reported to display nearly identical fragmentation patterns and chromatographic behavior. METHODS: Chromatographic separation of cyclopropylfentanyl and crotonylfentanyl by ultra-high-performance liquid chromatography was investigated using 3 different stationary phases (high strength silica T3, ethylsiloxane/silica hybrid C18, and Kinetex biphenyl) using gradient elution with a mobile phase consisting of 10 mM ammonium formate pH 3.1 and MeOH. Detection was performed by tandem mass spectrometry. In addition, the major metabolites of the 2 compounds formed on incubation with human liver microsomes were identified by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. RESULTS: Baseline separation of cyclopropylfentanyl and crotonylfentanyl was achieved on the ethylsiloxane/silica hybrid C18 column with retention times of 6.79 and 7.35 minutes, respectively. The major metabolites of the 2 analogues formed by human liver microsomes differed, with the main biotransformation being N-dealkylation and carboxylation for cyclopropylfentanyl and crotonylfentanyl, respectively. We demonstrated the usefulness of the 2 approaches by unambiguously identifying cyclopropylfentanyl, as well as its metabolites, in 2 authentic postmortem blood samples. CONCLUSIONS: In this study, we successfully demonstrated that cyclopropylfentanyl and crotonylfentanyl can be distinguished by methods commonly available in forensic laboratories.


Assuntos
Analgésicos Opioides/metabolismo , Fentanila/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Fentanila/metabolismo , Humanos , Laboratórios , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem/métodos
13.
Future Sci OA ; 5(1): FSO359, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30652024

RESUMO

Aim: For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared. Materials & methods: 'Case study' exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed. Results: Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC-MS. Western blotting and nanoLC-MS also revealed negative exosome markers regarding both isolation approaches. Conclusion: The two isolation methods had an overall similar performance, but we hesitate to use the term 'exosome isolation' as impurities may be present with both isolation methods. NanoLC-MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures.

14.
Ther Drug Monit ; 40(6): 738-748, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30157097

RESUMO

BACKGROUND: Fentanyl and fentanyl analogues (fentanyls) are very potent opioids posing a serious threat to the public health. Thousands of overdose deaths across the world are caused by fentanyls, and the numbers are increasing. Rapid mapping of current trends in opioid abuse is necessary to accelerate preventive measures. To ensure this, there is a need for sensitive targeted multiplex MS/MS methods to pinpoint drugs of abuse. We present a fully validated UHPLC-MS/MS method for the determination of 26 fentanyls, including several structural isomers, and the opioid antagonist naloxone in human whole blood. METHODS: Blood samples were prepared by liquid-liquid extraction with ethyl acetate and heptane. The fentanyls were separated with UHPLC, using a Kinetex biphenyl column (2.1 × 100 mm, 1.7 µm; Phenomenex, Verløse, Denmark) with an acidic mobile phase. Quantification was performed by MS/MS. The method was validated according to SWGTOX guidelines. RESULTS: The developed method could successfully separate all 27 analytes, including 7 isomers, and was validated according to SWGTOX guidelines with very low limits of quantification (4-20 pg/mL). The applicability of the method was demonstrated by determination of fentanyls in postmortem blood samples from 2 cases. CONCLUSIONS: A selective, highly sensitive, and robust method for determination of a large panel of fentanyls and naloxone in blood was developed and validated. Naloxone was included to monitor use and efficacy of the opioid antidote in cases of fentanyl overdoses. The method demonstrated good ability to separate structural isomers, which is important to differentiate between the numerous available fentanyls with variable potency, toxicity, and legal status. The developed method can be used to identify fentanyls on the drug market to help combat the fentanyl crisis.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fentanila/análogos & derivados , Fentanila/sangue , Espectrometria de Massas em Tandem/métodos , Analgésicos Opioides/sangue , Fentanila/química , Humanos , Extração Líquido-Líquido , Estrutura Molecular , Naloxona/sangue , Antagonistas de Entorpecentes/sangue , Detecção do Abuso de Substâncias/métodos
15.
J Chromatogr A ; 1534: 195-200, 2018 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-29290401

RESUMO

Open tubular liquid chromatography columns with organic polymer layers can be powerful tools for high sensitivity measurements in e.g. proteomics. However, these narrow columns are challenging to characterize. A two-electrode system, often used for bioimpendance measurements, was used to study poly(styrene-co-divinylbenzene = PS-DVB) polymer layered open tubular (PLOT) liquid chromatography columns with 10 µm inner diameters. The system performed electrical resistance measurements (ERM) for assessing layer thickness and porosity. Layer determination results were comparable (but more precise) to that obtained with scanning electron microscopy (SEM). Porosity examinations with ERM casted doubt on the presence/availability of pores in the layers investigated.


Assuntos
Cromatografia Líquida/métodos , Eletrodos , Microscopia Eletrônica de Varredura , Poliestirenos/química , Porosidade , Proteômica
16.
J Chromatogr A ; 1518: 104-110, 2017 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-28882340

RESUMO

An open tubular (OT) sample preparation/separation platform was developed. A multi-channel polymer layer open tubular (mPLOT) solid phase extraction (SPE) column was prepared by wall-coating the 126 channels (8µm inner diameter (ID) each) of a crystal fiber capillary with an organic polymer, namely poly(styrene-co-octadecene-co-divinylbenzene) (PS-OD-DVB). The mPLOT SPE was coupled on-line with a 10µm×2m poly(styrene-co-divinylbenzene) (PS-DVB) OT liquid chromatography column with nanospray mass spectrometry (OTLC-MS). Compared to using monolithic/particle-packed SPEs, mPLOT-SPE-OTLC allowed both fast loading and sufficient refocusing on the OT analytical column of small model compounds (sulfonamides≈300Da). Using automatic filtration/filter back-flushing (AFFL) plumbing, the mPLOT SPE column gave a constant and low back-pressure ≈35bar at 0.5µL/min. Surprisingly large sample volumes (10µL) were possible to be injected using a 12cm mPLOT.


Assuntos
Técnicas de Química Analítica/instrumentação , Cromatografia Líquida , Espectrometria de Massas , Extração em Fase Sólida/instrumentação , Poliestirenos/química , Polivinil/química
17.
Anal Chem ; 89(17): 8667-8673, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28783436

RESUMO

For counterterrorism purposes, a selective nano liquid chromatography-mass spectrometry (nanoLC-MS) platform was developed for detecting the highly lethal protein ricin from castor bean extract. Manual sample preparation steps were omitted by implementing a trypsin/Lys-C enzyme-immobilized multichannel reactor (MCR) consisting of 126 channels (8 µm inner diameter in all channels) that performed online digestion of proteins (5 min reaction time, instead of 4-16 h in previous in-solution methods). Reduction and alkylation steps were not required. The MCR allowed identification of ricin by signature peptides in all targeted mode injections performed, with a complete absence of carry-over in blank injections. The MCRs (interior volume ≈ 1 µL) have very low backpressure, allowing for trivial online coupling with commercial nanoLC-MS systems. The open tubular nature of the MCRs allowed for repeatable within/between-reactor preparation and performance.


Assuntos
Terrorismo Químico/prevenção & controle , Cromatografia Líquida/métodos , Ricina/análise , Espectrometria de Massas em Tandem/métodos , Reatores Biológicos , Semente de Rícino/química , Enzimas Imobilizadas/química , Metaloendopeptidases/química , Ricina/química , Ricina/isolamento & purificação , Tripsina/química
18.
Chem Phys Lipids ; 207(Pt B): 87-91, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28583433

RESUMO

Capillary electrophoresis (CE) can provide high separation efficiency with very simple instrumentation, but has yet to be explored regarding oxysterols/cholesterol. Cholesterol and 25-hydroxycholesterol (both are 4-ene-3-ketosteroids) were quantitatively transformed into hydrazones using Girard P reagent after enzymatic oxidation by cholesterol oxidase. Separation was achieved using non-aqueous capillary electrophoresis with UV detection at 280nm; the "charge-tagging" Girard P reagent ensured both charge and chromophore (which are requirements for CE-UV). Excess reagent was also separated from the two analytes, eliminating the need for removal prior to the analysis. The compounds were separated in less than 5min with excellent separation efficiency, using separation electrolytes fully compatible with mass spectrometry. The CE-UV method was used to optimize steps for charge-tagging, revealing that the procedure is affected by the analyte/reagent ratio and reaction time, but also the analyte structure.


Assuntos
Betaína/análogos & derivados , Colesterol/química , Colesterol/isolamento & purificação , Hidroxicolesteróis/química , Hidroxicolesteróis/isolamento & purificação , Betaína/química , Colesterol/metabolismo , Colesterol Oxidase/química , Colesterol Oxidase/metabolismo , Eletroforese Capilar , Hidroxicolesteróis/metabolismo , Conformação Molecular , Espectrofotometria Ultravioleta
19.
J Chromatogr A ; 1498: 111-119, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28385266

RESUMO

Self-preparation of nano liquid chromatography (nLC) columns has advantages regarding cost and flexibility. For targeted proteomics, we evaluated several approaches for particle-packing nLC columns and manufacturing fritless silica-based monolithic trap columns (50µm inner diameter). Our preferred approach for nLC column preparation was to magnetically stir Accucore core shell particles (C18 stationary phase) in ACN/water (80/20, v/v) suspensions during pressure-driven filling of polymer-fritted standard fused silica capillaries. The columns were ready for use about one hour after preparation had begun. They had comparable peak capacities (peptides) to commercial columns, and satisfactory within/between-column retention time repeatability, suited for targeted proteomics. Packing with commercial capillary housings/nanospray emitters did not improve performance compared to packing with in-house fritted stock fused silica capillary tubing. For trap columns, several recipes for narrow bore silica-based monolithic columns were evaluated, and we found the recipe by Zou et al. (2005) to be reproducible. Compared to the standard C18 trap column for Accucore nLC columns, monolith trap columns (C8 stationary phase) significantly reduced peak widths. The readily prepared in-house columns were used for targeted detection of the enzyme CYP27A1 in cancer cells, which is associated with proliferation and metastasis of breast cancer.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Nanopartículas/química , Peptídeos/análise , Proteômica/métodos , Dióxido de Silício/química , Linhagem Celular Tumoral , Colestanotriol 26-Mono-Oxigenase/análise , Colestanotriol 26-Mono-Oxigenase/isolamento & purificação , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Peptídeos/isolamento & purificação , Pressão
20.
J Clin Invest ; 127(2): 709-719, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-28112678

RESUMO

BACKGROUND: Sex, emotion, and reproduction are fundamental and tightly entwined aspects of human behavior. At a population level in humans, both the desire for sexual stimulation and the desire to bond with a partner are important precursors to reproduction. However, the relationships between these processes are incompletely understood. The limbic brain system has key roles in sexual and emotional behaviors, and is a likely candidate system for the integration of behavior with the hormonal reproductive axis. We investigated the effects of kisspeptin, a recently identified key reproductive hormone, on limbic brain activity and behavior. METHODS: Using a combination of functional neuroimaging and hormonal and psychometric analyses, we compared the effects of kisspeptin versus vehicle administration in 29 healthy heterosexual young men. RESULTS: We demonstrated that kisspeptin administration enhanced limbic brain activity specifically in response to sexual and couple-bonding stimuli. Furthermore, kisspeptin's enhancement of limbic brain structures correlated with psychometric measures of reward, drive, mood, and sexual aversion, providing functional significance. In addition, kisspeptin administration attenuated negative mood. CONCLUSIONS: Collectively, our data provide evidence of an undescribed role for kisspeptin in integrating sexual and emotional brain processing with reproduction in humans. These results have important implications for our understanding of reproductive biology and are highly relevant to the current pharmacological development of kisspeptin as a potential therapeutic agent for patients with common disorders of reproductive function. FUNDING: National Institute for Health Research (NIHR), Wellcome Trust (Ref 080268), and the Medical Research Council (MRC).


Assuntos
Emoções/efeitos dos fármacos , Kisspeptinas/administração & dosagem , Sistema Límbico/diagnóstico por imagem , Sistema Límbico/fisiologia , Comportamento Sexual/efeitos dos fármacos , Adulto , Método Duplo-Cego , Humanos , Masculino
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