RESUMEN
Hydrophilic interaction (liquid) chromatography (HILIC) has become the first choice LC mode for the separation of hydrophilic analytes. Numerous studies reported the poor retention time repeatability of HILIC. The problem was often ascribed to slow equilibration and insufficient re-equilibration time to establish the sensitive semi-immobilized water layer at the interface of the polar stationary phase and the bulk mobile phase. In this study, we compare retention time repeatability in HILIC for borosilicate glass and PFA (co-polymer of tetrafluoroethylene and perfluoroalkoxyethylene) solvent bottles. During this study, we observed peak patterns shifting towards higher retention times (for metabolites and peptides) and lower retention times (oligonucleotide sample) with ongoing analysis time when standard borosilicate glass bottles were used as solvent reservoirs. It was hypothesized that release of ions (sodium, potassium, borate, etc.) from the borosilicate glass bottles leads to alterations (thickness and electrostatic screening effects) in the semi-immobilized water layer which is adsorbed to the polar stationary phase surface under acetonitrile-rich eluents in HILIC with concomitant shifts in retention. When PFA solvent bottles were employed instead of borosilicate glass, retention time repeatability was greatly improved and changed from average 8.4 % RSD for the tested metabolites with borosilicate glass bottles to 0.14 % RSD for the PFA solvent bottles (30 injections over 12 h). Similar improvements were observed for peptides and oligonucleotides. This simple solution to the retention time repeatability problem in HILIC might contribute to a better acceptance of HILIC, especially in fields like targeted and untargeted metabolomics, peptide and oligonucleotide analysis.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Cromatografía Liquida/métodos , Reproducibilidad de los Resultados , Péptidos/análisis , Péptidos/química , Fluorocarburos/análisis , Fluorocarburos/química , Oligonucleótidos/análisis , Oligonucleótidos/química , Solventes/química , Vidrio/químicaRESUMEN
Alterations in the function of K+ channels such as the voltage- and Ca2+-activated K+ channel of large conductance (BKCa) reportedly promote breast cancer (BC) development and progression. Underlying molecular mechanisms remain, however, elusive. Here, we provide electrophysiological evidence for a BKCa splice variant localized to the inner mitochondrial membrane of murine and human BC cells (mitoBKCa). Through a combination of genetic knockdown and knockout along with a cell permeable BKCa channel blocker, we show that mitoBKCa modulates overall cellular and mitochondrial energy production, and mediates the metabolic rewiring referred to as the 'Warburg effect', thereby promoting BC cell proliferation in the presence and absence of oxygen. Additionally, we detect mitoBKCa and BKCa transcripts in low or high abundance, respectively, in clinical BC specimens. Together, our results emphasize, that targeting mitoBKCa could represent a treatment strategy for selected BC patients in future.
Asunto(s)
Neoplasias de la Mama , Humanos , Animales , Ratones , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Mitocondrias/metabolismo , Mitocondrias/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Membranas Mitocondriales/metabolismo , Femenino , Metabolismo EnergéticoRESUMEN
Glutathione, its biosynthesis intermediates, and other thiol metabolites are of central relevance for the redox homeostasis of cells. Their analysis is critical due to the facile interconversion of redox pairs during sampling, sample preparation, and data acquisition, in particular in the electrospray ionization interface. In this work, we propose a fast-targeted liquid chromatography-tandem mass spectrometry method to accurately analyze 14 metabolites from the glutathione pathway. N-Ethylmaleimide reagent is added with the extraction solvent and instantly stabilizes the thiol-redox state by derivatization. Liquid chromatographic separation of the analytes was performed on a sub-2 µm superficially porous hydrophilic interaction liquid chromatography column with sulfobetaine chemistry. Tandem mass spectrometry with triple-quadrupole mass spectrometry in multiple-reaction monitoring acquisition mode allowed sensitive detection of the targeted metabolites with limits of quantification in the range of 5-25 nM. Run times of 3 min enable a high throughput analysis of cellular samples. For calibration, a 13 C-labelled cell extract was used as an internal standard. The method was validated and the concentrations of glutathione and its biosynthesis intermediates were determined in HeLa cells.
Asunto(s)
Compuestos de Sulfhidrilo , Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Células HeLa , Cromatografía Liquida/métodos , Cromatografía Líquida de Alta Presión/métodosRESUMEN
In this study, the chromatographic behavior of mixed-mode and hydrophilic interaction liquid chromatography (HILIC) with the mixed-mode HILIC/strong anion-exchange (SAX) column HILICpak VT-50 2D and the two HILIC columns Atlantis Premier BEH Z-HILIC and Acquity Premier BEH Amide was assessed with regard to their separation capability of the metabolites from the glycolysis and pentose phosphate pathways. Chromatographic conditions were evaluated with the aim of achieving separation of the isomeric glycolytic phosphorylated carbohydrate metabolites free from isomeric interferences and thus allowing for selective targeted analysis by liquid chromatography with tandem mass spectrometry (MS/MS) using multiple reaction monitoring acquisition. The effects of pH values (8.0/9.0/10.0) of the ammonium bicarbonate buffer and gradient time were investigated during HILIC-MS/MS analysis, with the optimal conditions found at pH = 10.0. Separation of the pentose phosphate isomers (ribose 5- and 1-phosphate, xylulose 5-phosphate and ribulose 5-phosphate) was achieved on the mixed-mode HILIC/SAX (HILICpak VT-50 2D) column and HILIC BEH Amide column. Column performance was evaluated based on the direct comparison of chromatographic parameters, i.e. peak width at 50% and peak tailing factors of the individual metabolites. Parity plots were generated allowing a direct comparison between the normalized retention times and assessment of orthogonality of all 3 stationary phases evaluated. Separation of 7 biologically relevant hexose monophosphates metabolites turned out to be challenging by HILIC-MS/MS, with the BEH Amide providing the best individual results for such a separation. However, fructose 6-phosphate and glucose 1-phosphate co-eluted. Therefore, an on-line heart-cutting HILIC-Mixed Mode 2D-LC-QToF experiment was conducted, allowing the separation of this critical isomer pair. In this setup, the BEH Amide column in the 1D separated the majority of target metabolites, while a heart-cut of the peak from totally coeluted fructose 6-phosphate and glucose 1-phosphate was separated in the 2D with HILICpak VT50-2D column, thus allowing undisturbed determination of the glycolytic phosphorylated carbohydrate metabolites due to their chromatographic separation from hexose monophosphate metabolites. The assay specificity towards 7 common hexose monophosphates was characterized (glucose 1- and 6-phosphate, galactose 1- and 6-phosphate, fructose 6-phosphate, mannose 1- and 6-phosphate). The selectivity of some rare hexose monophosphates (allose 6-phosphate, tagatose 6-phosphate, sorbose 1-phosphate) was also tested.
Asunto(s)
Fosfatos de Azúcar , Espectrometría de Masas en Tándem , Vía de Pentosa Fosfato , Cromatografía Liquida/métodos , Carbohidratos , Glucosa , Interacciones Hidrofóbicas e Hidrofílicas , Amidas , Fosfatos , FructosaRESUMEN
This work reports on targeted UHPLC-tandem mass spectrometry methods for the chiral separation of anteiso-methyl branched fatty acids (aiFAs). The methods involve precolumn derivatization with 1-naphthylamine and chiral separation on Chiralpak IG-U. anteiso-Methyl branched fatty acids with up to eight carbons can be separated. A method was used for the assignment of the absolute configuration of an aiFA present as fatty acyl residue of the teicoplanin mixture, namely teicoplanin RS3. Furthermore, the excellent methylene selectivity and improved selectivity for constitutional isomers of the polysaccharide columns was exploited for the elucidation and structural confirmation of previously unknown fatty acyl residues in teicoplanin. This shows the versatility and practical applicability of polysaccharide columns as orthogonal stationary phases to reversed-phase for structural elucidation of natural compounds. The developed methods are useful tools for related subdisciplines such as targeted metabolomics and lipidomics.
Asunto(s)
Espectrometría de Masas en Tándem , Teicoplanina , Teicoplanina/química , Cromatografía Líquida de Alta Presión/métodos , Ácidos Grasos , Polisacáridos , EstereoisomerismoRESUMEN
In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. MRM ion pairs were acquired from HeLa cell samples through untargeted analysis using UHPLC-QTOF-MS with SWATH acquisition complemented by missing metabolites from pathway databases. Four different cell extraction protocols were studied and compared based on an experiment series involving the calculation of individual metabolite recoveries (pre/post extraction spiking U-13C isotope-labeled standards), with a Methanol/Water extraction mixture (1:1; v/v) showing the best results. Two HILIC-MS methods employing a Waters Premier BEH Amide column were developed, utilizing two different chromatographic conditions (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI+ mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI- mode. One hundred sixty-one (161) metabolites were successfully detected in ESI+ mode, whereas 92 were detected in negative ionization mode, totaling to a number of 253 compounds in three different biological matrices covered by the analytical system employed. Both established HILIC methods were calibrated and validated based on 105 authentic chemical standards and U-13C-labeled Pichia pastoris (Komagataella phaffii) yeast extract as internal standards for cellular matrix (HeLa cells). Within-day and between-day precision was determined on three different QC concentration levels and was below 15% for the entirety of the analytes. Inter- and intra-day accuracies showed values in the range between 85 and 115% (assessed as % recovery) in the entire range. Matrix effects, extraction recoveries and process efficiencies were evaluated following the Matuszewski protocol with U-13C-labeled Pichia pastoris metabolite extract as internal standards. Eventually, the method was utilized to quantify metabolites in HeLa cell extracts.
Asunto(s)
Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Extractos Celulares , Células HeLa , Flujo de Trabajo , Cromatografía Liquida/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Cromatografía Líquida de Alta Presión/métodosRESUMEN
Branched-chain fatty acids (BCFAs) are mostly saturated fatty acids with one or more methyl, seldom ethyl, branches in the alkyl chain. They are derived from branched-chain amino acids, ruminant-derived food, or biosynthetic side products of acetyl-CoA carboxylase. They possess iso- (branching at penultimate carbon) and anteiso-fatty acid structure (branching at antepenultimate carbon) or are branched at any other position of the carbon chain. Except for iso-fatty acids, BCFAs are chiral. They are commonly analyzed by GC-MS, while there is a lack of enantioselective LC-MS methods. In this work, we present a methodology for targeted enantioselective UHPLC-ESI-MS/MS metabolomics of BCFAs. It makes use of precolumn derivatization with 1-naphthylamine and reversed-phase elution conditions. A homologous series of short BCFA analytes with distinct chain lengths (having up to eight carbon atoms), branching type (methyl or ethyl), and position of branching (2, 3, and 4, anteiso and iso) has been systematically studied on six commercially available polysaccharide UHPLC columns. Chiralpak IB-U exhibited the highest and broadest enantioselectivity while IH-U maintained enantioselectivity also for BCFAs with chirality distant from the carboxylic function (i.e., with other branching than in 2-position). The method was used to assign the absolute configuration of a 4-methylhexanoic acid side chain of a natural product from Streptomyces sp. SHP 22-7. The potential of the corresponding UHPLC-ESI-QTOF-MS/MS assay for analyzing stereoselectively BCFAs and other short organic acids by untargeted analysis in human urine was further elucidated in a preliminary proof-of-principle test.