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1.
Anal Bioanal Chem ; 413(11): 2971-2984, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33693976

RESUMEN

The kallikrein-kinin system (KKS) is involved in many physiological and pathophysiological processes and is assumed to be connected to the development of clinical symptoms of angioedema or COVID-19, among other diseases. However, despite its diverse role in the regulation of physiological and pathophysiological functions, knowledge about the KKS in vivo remains limited. The short half-lives of kinins, their low abundance and structural similarities and the artificial generation of the kinin bradykinin greatly hinder reliable and accurate determination of kinin levels in plasma. To address these issues, a sensitive LC-MS/MS platform for the comprehensive and simultaneous determination of the four active kinins bradykinin, kallidin, des-Arg(9)-bradykinin and des-Arg(10)-kallidin and their major metabolites bradykinin 2-9, bradykinin 1-7 and bradykinin 1-5 was developed. This platform was validated according to the bioanalytical guideline of the US Food and Drug Administration regarding linearity, accuracy, precision, sensitivity, carry-over, recovery, parallelism, matrix effects and stability in plasma of healthy volunteers. The validated platform encompassed a broad calibration curve range from 2.0-15.3 pg/mL (depending on the kinin) up to 1000 pg/mL, covering the expected concentrations in disease states. No source-dependent matrix effects were identified, and suitable stability of the analytes in plasma was observed. The applicability of the developed platform was proven by the determination of endogenous levels in healthy volunteers, whose plasma kinin levels were successfully detected in the low pg/mL range. The established platform facilitates the investigation of kinin-mediated diseases (e.g. angioedema, COVID-19) and enables the assessment of the impact of altered enzyme activities on the formation or degradation of kinins.


Asunto(s)
Bradiquinina/análogos & derivados , Bradiquinina/sangre , Calidina/análogos & derivados , Calidina/sangre , Sistema Calicreína-Quinina , Espectrometría de Masas en Tándem/métodos , /sangre , Cromatografía Liquida/métodos , Humanos , Límite de Detección , Fragmentos de Péptidos/sangre
2.
J Chromatogr A ; 1642: 462045, 2021 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-33735643

RESUMEN

A convenient synthetic approach to a linear alkyl-polyamine amphiphilic chromatographic selector was proposed. Successive immobilization of the amphiphile onto silica gel afforded a multimodal stationary phase for high-performance liquid chromatography (HPLC). The as-prepared silica material was studied comparatively with a conventional octadecyl (C18) and an amide-embedded C18 stationary phase. The new uniform docosyl-triamine tandem was featured by an enhanced shape selectivity towards geometric isomers, and a low silanol activity towards alkaline solutes. The presence of multiple amino groups rendered the new adsorbent operable in different modes, such as hydrophilic interaction and ion-exchange modes. The satisfactory performance of the said stationary phase in separating different classes of analytes, including polycyclic aromatic hydrocarbons, flavonoids, tricyclic antidepressants, calcium channel blockers, aromatic acids, inorganic anions, nucleosides and estrogens, revealed its great potential and high adaptability for multipurpose LC utility.


Asunto(s)
Cromatografía Liquida/métodos , Poliaminas/química , Tensoactivos/química , Estrógenos/análisis , Flavonoides/análisis , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Poliaminas/síntesis química
3.
J Chromatogr A ; 1642: 462047, 2021 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-33744605

RESUMEN

As the reliance on metabolic biomarkers within drug discovery and development increases, there is also an increased demand for global metabolomics methods to provide broad metabolome coverage and sensitivity towards differences in metabolite expression and reproducibility. A systematic approach is necessary for the development, and evaluation, of metabolomics methods using either conventional techniques or when establishing new methods that allow for additional gains in sensitivity and a reduction in requirements for amounts of a biological sample, such as those seen with methods based on microseparations. We developed a novel standard mixture and used a systematic approach for the development and optimization of optimal, ion-pair free, liquid chromatography-mass spectrometry (LC-MS) global profiling methods. These methods were scaled-down to microflow-based LC separations and compared with analytical flow ion-pairing reagent containing methods. Average peak volume improvements of 7- and 22-fold were observed in the positive and negative ionization mode microflow methods as compared to the ion-pairing reagent analytical flow methods, respectively. The linear range of the newly developed microflow methods showed up to a 10-fold increase in the lower limit of detection in the negative ionization mode. The developed microflow LC-MS methods were further evaluated using wild-type mouse plasma where up to a 9-fold increase in peak volume was observed.


Asunto(s)
Cromatografía Liquida/métodos , Desarrollo de Medicamentos , Descubrimiento de Drogas , Espectrometría de Masas/métodos , Metabolómica , Reología , Animales , Humanos , Metaboloma , Ratones , Estándares de Referencia , Reproducibilidad de los Resultados
4.
J Chromatogr A ; 1642: 462030, 2021 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-33721812

RESUMEN

The total solute retention by a chemically modified stationary phase (CMSP) has been shown several times to be a potential tool for studying the binding abilities of the bound compound. In this article, we present a methodology for the deconvolution of the total retention into structure-specific contributions. Three complementary silica-based CMSPs were prepared: 1) non-modified silica, 2) silica modified by syn-bis-Tröger's base (a molecular tweezer) and 3) silica modified by anti-bis-Tröger's base (a non-tweezer molecule). These were characterized by elemental analysis and Raman spectroscopy, and used to assemble liquid chromatography (LC) columns. The total retention factors were estimated for electron-deficient nitro- and cyano-derivatives of benzene in both normal and reverse elution modes. The total retention factor was considered to be the sum of structure-specific retention factors, each related to the affinity (the binding constant) of a specific structure (the binding site), and its content in the modified silica, as defined for weak-affinity chromatography (WAC). The obtained structure-specific contributions are in line with the binding studies of ligands in solution. They reveal details of the retention mechanism, suggesting a more suitable attachment of ligands, and expose the shortcomings of evaluations based solely on the total retentions.


Asunto(s)
Cromatografía Liquida/métodos , Dióxido de Silicio/química , Ligandos , Soluciones , Estereoisomerismo
5.
Arch Insect Biochem Physiol ; 106(4): e21778, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33719129

RESUMEN

One representative of the order Trichoptera, namely the caddisfly Chaetopteryx villosa, was investigated along with the pygmy mole cricket Xya capensis which is a representative of the most basal superfamily of the caeliferan Orthoptera, that is, the Tridactyloidea. From both clades neuropeptides have not been biochemically characterized before this study. Here, members of the adipokinetic hormone family (AKHs) are sequenced via liquid chromatography (LC)-ion trap mass spectrometry from methanolic extracts from the corpora cardiaca of respective species. The corpora cardiaca were dissected, methanolic extracts prepared, peptides separated by liquid chromatography (LC), and AKHs detected and sequenced by ion trap mass spectrometry. Both species investigated contain an octapeptide AKH: the trichopteran species has the peptide with the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Ser-Trp amide; the ambiguity of the isobaric amino acids Leu and Ile at position two was solved by comparing retention times on LC and by co-elution with the synthetic Leu2 -form. This peptide is known as Aedae-AKH and found in certain dipteran species and in an alderfly (Megaloptera). The tridactyloid species contains the peptide with the sequence pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp amide which had first been identified in a member of the order Mantophasmatodea and is called Manto-CC. Comparisons are made between the AKH complements of the sister groups Trichoptera and Lepidoptera and their possible relatedness and, on the other hand, between the AKH of X. capensis with those of closely related caeliferan superfamilies. The biology of the two studied species is used to speculate about a possible function of the elucidated hormones. Lastly, the use of a larval stage as starting material for structural neuropeptide information is discussed.


Asunto(s)
Gryllidae/metabolismo , Insectos/metabolismo , Neuropéptidos , Animales , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Neuropéptidos/química , Neuropéptidos/aislamiento & purificación , Neuropéptidos/metabolismo
6.
Methods Mol Biol ; 2259: 3-12, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687705

RESUMEN

In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVs.


Asunto(s)
Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestructura , Células Madre Mesenquimatosas/citología , Proteínas/análisis , Células Cultivadas , Cromatografía Liquida/métodos , Medios de Cultivo/química , Humanos , Células Madre Mesenquimatosas/química , Microscopía Electrónica de Transmisión/métodos , Cultivo Primario de Células/métodos , Proteínas/aislamiento & purificación , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Cordón Umbilical/citología
7.
Methods Mol Biol ; 2259: 59-75, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687709

RESUMEN

Anisakis simplex s.s. is a parasitic nematode that causes anisakiasis in humans. L3 stage larvae, which are present in many fish species and cephalopods all over the globe, might be consumed and develop occasionally into the L4 stage but cannot reproduce. Anisakiasis is an emerging health problem and economic concern. In recent years, proteomic methods have gained greater acceptance among scientists involved in parasitology and food science. According to that, here, we present tandem mass tag (TMT)-based shotgun proteomics to define differences in proteomic composition between L3 and L4 development stages of A. simplex s.s.


Asunto(s)
Anisakis/crecimiento & desarrollo , Proteínas del Helminto/análisis , Proteómica/métodos , Animales , Anisakiasis/parasitología , Anisakis/química , Anisakis/metabolismo , Cromatografía Liquida/métodos , Proteínas del Helminto/metabolismo , Humanos , Larva/química , Larva/crecimiento & desarrollo , Larva/metabolismo , Espectrometría de Masas en Tándem/métodos
8.
Methods Mol Biol ; 2259: 167-179, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687714

RESUMEN

Metaproteomics of host-microbiome interfaces comprises the analysis of complex mixtures of bacteria, archaea, fungi, and viruses in combination with its host cells. Microbial niches can be found all over the host including the skin, oral cavity, and the intestine and are considered to be essential for the homeostasis. The complex interactions between the host and diverse commensal microbiota are poorly characterized while of great interest as dysbiosis is associated with the development of various inflammatory and metabolic diseases. The metaproteomics workflows to study these interfaces are currently being established, and many challenges remain. The major challenge is the large diversity in species composition that make up the microbiota, which results in complex samples that require extended mass spectrometry analysis time. In addition, current database search strategies are not developed to the size of the search space required for unbiased microbial protein identification.Here, we describe a workflow for the proteomics analysis of microbial niches with a focus on intestinal mucus layer. We will cover step-by-step the sample collection, sample preparation, liquid chromatography-mass spectrometry, and data analysis.


Asunto(s)
Bacterias/aislamiento & purificación , Proteínas Bacterianas/análisis , Proteínas Fúngicas/análisis , Hongos/aislamiento & purificación , Microbioma Gastrointestinal , Proteómica/métodos , Animales , Cromatografía Liquida/métodos , Intestinos/microbiología , Espectrometría de Masas/métodos , Ratones , Péptidos/análisis , Flujo de Trabajo
9.
Methods Mol Biol ; 2259: 181-189, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687715

RESUMEN

Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."


Asunto(s)
Proteínas Bacterianas/análisis , Mycobacterium/química , Proteómica/métodos , Cromatografía Liquida/métodos , Humanos , Mycobacterium/citología , Infecciones por Mycobacterium/microbiología , Mycobacterium smegmatis/química , Mycobacterium smegmatis/citología , Proteoma/análisis , Espectrometría de Masas en Tándem/métodos
10.
Methods Mol Biol ; 2259: 191-201, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687716

RESUMEN

Recent advances in MS/MS technology have made it possible to use proteomic data to predict protein-coding sequences. This approach is called proteogenomics, and it allows to correctly translate start and stop sites and to reveal new open reading frames. Here, we focus on using proteogenomics to improve the annotation of Mycobacteriumtuberculosis strains. We also describe detail procedures of the extraction of proteins and their further preparation for LC-MS/MS analysis and outline the main steps of data analysis.


Asunto(s)
Proteínas Bacterianas/genética , Mycobacterium tuberculosis/genética , Proteogenómica/métodos , Tuberculosis/microbiología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/aislamiento & purificación , Cromatografía Liquida/métodos , Genoma Bacteriano , Humanos , Anotación de Secuencia Molecular , Mycobacterium tuberculosis/química , Espectrometría de Masas en Tándem/métodos
11.
Methods Mol Biol ; 2259: 205-213, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687717

RESUMEN

Classical and culture-based methods for the identification and characterization of the biochemical properties of microorganisms are slow and labor-intensive. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been used for the analysis of bacterial pathogen strain-specific diagnostic peptides allowing the characterization of bacterial strains.Here, we describe the analysis of tryptic digestion peptides by LC-ESI-MS/MS to search for specific biomarkers useful for the rapid identification of, on the one hand, the bacterial species and, on the other hand, the physiological and biochemical characteristics such as the expression of virulence factors, including toxins, immune-modulatory factors, and exoenzymes.


Asunto(s)
Bacterias/aislamiento & purificación , Proteínas Bacterianas/análisis , Microbiología de Alimentos , Proteómica/métodos , Proteínas Bacterianas/aislamiento & purificación , Cromatografía Liquida/métodos , Programas Informáticos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos
12.
Methods Mol Biol ; 2259: 215-223, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687718

RESUMEN

A workflow for the characterization of food-derived bioactive peptides is described in this chapter. The workflow integrates two consecutive steps: a discovery phase and a protein-based bioinformatic phase. In the first step (discovery phase), a shotgun bottom-up proteomics approach is used to create a reference data set for a selected food proteome. Afterward, in a second step (bioinformatic phase), the reference proteome is subjected to several in silico protein-based bioinformatic analyses to predict and characterize potential bioactive peptides after an in silico human gastrointestinal digestion. Using this workflow, bioactive collagen peptides, antihypertensive, antimicrobial, and antitumor peptides were predicted as potential valuable bioactive peptides from seafood and marine by-products. It is concluded that the combination of the global shotgun proteomic analysis and the analysis by protein-based bioinformatics can provide a rapid strategy for the characterization of new potential food-derived bioactive peptides.


Asunto(s)
Proteínas en la Dieta/análisis , Péptidos/análisis , Proteómica/métodos , Cromatografía Liquida/métodos , Alimentos Funcionales/análisis , Humanos , Alimentos Marinos/análisis , Espectrometría de Masas en Tándem/métodos
13.
Methods Mol Biol ; 2259: 227-246, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687719

RESUMEN

Carbonylation is a nonenzymatic irreversible posttranslational protein modification and the main hallmark of protein oxidative damage. Elevated levels of protein carbonyl groups have been detected in age-related and metabolic diseases such as obesity, diabetes, Alzheimer, Parkinson, and several other oxidative stress-related maladies. Interestingly, many studies have shown that only a subset of proteins is carbonylated under the conditions of oxidative stress, demonstrating that carbonylation is a highly selective process. As a consequence, identifying and quantifying the disease-induced changes on a certain carbonylome are crucial to understanding the etiology and progression of numerous diseases and then designing adequate prevention/palliation strategies. However, the low abundance of carbonylated proteins in vivo, the enormous diversity of reactive species, and their relative lability make the analysis of carbonylated proteins a challenging task for redox proteomic technology. Therefore, we present a proteomic approach based on the labeling of carbonyls formed in vivo on proteins using the fluorescein 5-thiosemicarbazide (FTSC) tag to detect the subset of carbonylated proteins among a complex mixture of proteins regardless of the nature of carbonyl adduct, isolation and relative quantification of carbonylated proteins in 2D gel electrophoresis, and protein identification by LC-MS/MS analysis. This method has been successfully used for the evaluation of in vivo protein carbonylation in very diverse animal tissues (plasma, liver, kidney, skeletal muscle, and adipose tissue) and species (from fish to mammalian) and has also been applied in different research fields (from food technology to nutrition), demonstrating its robustness and reliability.


Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Carbonilación Proteica , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Liquida/métodos , Fluoresceínas/química , Colorantes Fluorescentes/química , Humanos , Focalización Isoeléctrica/métodos , Oxidación-Reducción , Proteoma/análisis
14.
Methods Mol Biol ; 2259: 297-308, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687723

RESUMEN

Shotgun proteomics is the inferential analysis of proteoforms using peptide proxies produced by enzyme-catalyzed hydrolysis of entire proteomes. Such peptides are usually identified by nanoflow liquid chromatography coupled to tandem mass spectrometry analysis (nLC-MS/MS). Traditionally, MS/MS analysis is performed in data-dependent acquisition (DDA) mode, which usually produces a pattern of fragment masses unique to a single peptide's fragmentation. Here, I describe a statistically rigorous qualitative and quantitative computational analysis for shotgun proteomics DDA analysis using free open-source software tools. MS/MS data are used to identify peptides, and the area of peptide mass/charge over chromatographic elution is used to quantify peptides. All peptides that uniquely map to a protein sequence predicted from the genome are combined into a single protein quantity, which can then be compared across experimental conditions. Statistically significant protein changes can be summarized using gene ontology or pathway term enrichment analysis.


Asunto(s)
Fragmentos de Péptidos/análisis , Proteínas/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Liquida/métodos , Ontología de Genes , Humanos , Anotación de Secuencia Molecular , Proteoma/análisis , Programas Informáticos
15.
Nat Commun ; 12(1): 1897, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772030

RESUMEN

Oxidative damage to DNA generates 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as two major lesions. Despite the comparable prevalence of these lesions, the biological effects of oxoA remain poorly characterized. Here we report the discovery of a class of DNA interstrand cross-links (ICLs) involving oxidized nucleobases. Under oxidative conditions, oxoA, but not oxoG, readily reacts with an opposite base to produce ICLs, highlighting a latent alkylating nature of oxoA. Reactive halogen species, one-electron oxidants, and the myeloperoxidase/H2O2/Cl- system induce oxoA ICLs, suggesting that oxoA-mediated cross-links may arise endogenously. Nucleobase analog studies suggest C2-oxoA is covalently linked to N2-guanine and N3-adenine for the oxoA-G and oxoA-A ICLs, respectively. The oxoA ICLs presumably form via the oxidative activation of oxoA followed by the nucleophilic attack by an opposite base. Our findings provide insights into oxoA-mediated mutagenesis and contribute towards investigations of oxidative stress-induced ICLs and oxoA-based latent alkylating agents.


Asunto(s)
Adenina/análogos & derivados , Daño del ADN , ADN/química , Estrés Oxidativo , Adenina/química , Cromatografía Liquida/métodos , Reactivos de Enlaces Cruzados/química , ADN/genética , ADN/metabolismo , Reparación del ADN , Guanina/análogos & derivados , Guanina/química , Espectrometría de Masas/métodos , Modelos Químicos , Estructura Molecular , Oxidación-Reducción
16.
Methods Mol Biol ; 2259: 247-257, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687720

RESUMEN

Protein phosphorylation is a critical posttranslational modification (PTM), with cell signaling networks being tightly regulated by protein phosphorylation. Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides that often have multiple phosphorylation sites. Herein, we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method, we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor (GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This protocol is easy to adopt and implement and thus should have broad utility for effective RPLC-MS analysis of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.


Asunto(s)
Fosfopéptidos/análisis , Proteómica/métodos , Cromatografía Liquida/métodos , Cromatografía de Fase Inversa/métodos , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoprecipitación/métodos , Células MCF-7 , Fosfopéptidos/aislamiento & purificación , Proteoma/análisis , Proteoma/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos
17.
Int J Mol Sci ; 22(4)2021 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-33567512

RESUMEN

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17ß-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.


Asunto(s)
Bioensayo/métodos , Biomarcadores/metabolismo , Disruptores Endocrinos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteoma/análisis , Espectrometría de Masas en Tándem/métodos , Útero/metabolismo , Animales , Biomarcadores/análisis , Cromatografía Liquida/métodos , Femenino , Ratas , Ratas Sprague-Dawley , Útero/efectos de los fármacos
18.
J Chromatogr A ; 1641: 461989, 2021 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-33611115

RESUMEN

Open tubular liquid chromatography (OT-LC) can provide superior chromatographic performance and more favorable mass spectrometry (MS) detection conditions. These features could provide enhanced sensitivity when coupled with electrospray ionization sources (ESI-) and lead to unprecedented detection capabilities if interfaced with a highly structural informative electron ionization (EI) source. In the past, the exploitation of OT columns in liquid chromatography evolved slowly. However, the recent instrumental developments in capillary/nanoLC-MS created new opportunities in developing and applying OT-LC-MS. Currently, the analytical advantages of OT-LC-MS are mainly exploited in the fields of proteomics and biosciences analysis. Nevertheless, under the right conditions, OT-LC-MS can also offer superior chromatographic performance and enhanced sensitivity in analyzing small molecules. This review will provide an overview of the latest developments in OT-LC-MS, focusing on the wide variety of employed separation mechanisms, innovative stationary phases, emerging column fabrication technologies, and new OT formats. In the same way, the OT-LC's opportunities and shortcomings coupled to both ESI and EI will be discussed, highlighting the complementary character of those two ionization modes to expand the LC's detection boundaries in the performance of targeted and untargeted studies.


Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas , Escherichia coli/metabolismo , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray
19.
J Chromatogr A ; 1641: 461965, 2021 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-33611125

RESUMEN

The injection volume and the associated column volume overload is one of the most common issues in miniaturized chromatography. The injection volume should not exceed 10% of the effective column volume. A further reduction of the injection volume leads to an increase in chromatographic efficiency. However, the signal intensity must be above a certain threshold to generate a chromatographic peak that can be detected. Therefore, the injection volume has to be optimized to reach the ideal balance between chromatographic efficiency and sensitivity. This study examined the general influence of the injection volume for both isocratic and gradient elution, depending on the retention factor and peak standard deviation. For this purpose, substances of different polarity were selected to represent a broad elution spectrum. Besides the model analyte naphthalene, these were mainly pharmaceuticals. For all measurements a microbore column with an ID of 300 µm and packed with 1.9 µm fully porous particles was used. For isocratic elution, the injection volume was varied between 4 and 16% of the effective column volume. The retention factors were adjusted between 2 and 10. For gradient elution, the injection volume was varied between 4 and 160% of the effective column volume. The observed effects were further investigated using the gradient kinetic plot theory. In isocratic elution, a loss in plate height up to 50% was observed for components that elute near the void time. A significant reduction of the chromatographic efficiency was noticed up to a retention factor of 4. In gradient elution, a reduction in peak capacity could only be observed if the injection volume exceeded 40% of the effective column volume. For some substances, only a slight loss in peak capacity was noticed even with a volume overload of 160%.


Asunto(s)
Cromatografía Liquida/métodos , Cinética , Metronidazol/análisis , Naftalenos/análisis , Porosidad , Estándares de Referencia
20.
J Chromatogr A ; 1639: 461932, 2021 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-33535117

RESUMEN

Position-specific isotope analysis by Nuclear Magnetic Resonance spectrometry was employed to study the 13C intramolecular isotopic fractionation associated with the migration of organic substrates through different stationary phases chromatography columns. Liquid chromatography is often used to isolate compounds prior to their isotope analysis and this purification step potentially alters the isotopic composition of target compounds introducing a bias in the later measured data. Moreover, results from liquid chromatography can yield the sorption parameters needed in reactive transport models that predict the transport and fate of organic contaminants to in the environment. The aim of this study was to use intramolecular isotope analysis to study both 13C and 15N isotope effects associated with the elution of paracetamol (acetaminophen) through different stationary phases and to compare them to effects observed previously for vanillin. Results showed very different intramolecular isotope fractionation profiles depending on the chemical structure of the stationary phase. The data also demonstrate that both the amplitude and the distribution of measured isotope effects depend on the nature of the non-covalent interactions involved in the migration process. Results provided by theoretical calculation performed during this study also confirmed the direct link between observed intramolecular isotope fractionation and the nature of involved intermolecular interactions. It is concluded that the nature of the stationary phase through which the substrate passes has a major impact on the intramolecular isotopic composition of organic compounds isolated by chromatography methods..


Asunto(s)
Acetaminofén/análisis , Isótopos de Carbono/química , Cromatografía Liquida/métodos , Isótopos de Nitrógeno/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Celulosa/química , Carbón Orgánico/química , Fraccionamiento Químico , Reproducibilidad de los Resultados , Gel de Sílice/química , Solventes/química
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