RESUMO
Polypropylene microcentrifuge tubes (MCTs) are increasingly used in lipidome sample preparation. In the absence of a comprehensive study evaluating ramifications of plasticware utilization in mass spectrometry-based lipidomic analyses, we conducted a systematic analysis to elucidate potential negative effects ascribable to labware contamination in serum lipidomics. During serum lipid extractions, tested glassware introduced 24 labware contaminants. In contrast, Eppendorf polypropylene MCTs contributed 485 contaminant features, many of which could be erroneously putatively identified as lipids via their m/z values. Eppendorf MCTs contamination engendered severe ion-suppression of 40 low abundance serum lipids, while generating mild to modest lipid ion-suppression across a multitude of higher abundance coeluting lipids. Less compatible polypropylene MCTs from an alternative manufacturer introduced a staggering 2,949 contaminant m/z values, severely affecting 75 coeluting serum lipids and causing more frequent and pronounced ion-suppression instances. Furthermore, by performing serum extractions with varied initial volumes, it was ascertained that labware-induced lipid ion-suppression is a dynamic phenomenon, contingent on both lipid and labware contaminant concentrations where low-abundance lipids are disproportionately impacted by coelutes of suppressive contaminants. In addition to lipid ion-suppression, the identification and quantification of 7 fatty acid endogenous serum lipids were compromised by the leaching of structurally identical surfactants from MCTs. MCTs artificially introduced 10 additional primary amides extraneous to serum samples. Utmost caution is imperative in interpreting data concerning primary amides and fatty acids when employing plastic labware. Through this investigation, we aspire to elevate awareness regarding the pernicious impact of labware contamination on lipidome analysis.
Assuntos
Lipidômica , Lipídeos , Espectrometria de Massas , Polipropilenos , Humanos , Lipidômica/métodos , Lipídeos/sangue , Lipídeos/química , Espectrometria de Massas/métodos , Polipropilenos/química , Contaminação de EquipamentosRESUMO
In lipidomic analysis, plasticware is increasingly being used for lipid extraction and other sample processing procedures over glassware. However, a systematic investigation of the consequences of plasticware use on mass spectrometry (MS)-based lipidome analysis is lacking. In this work, we present an analytical approach for detecting and comparing solvent and labware contaminants encountered in lipidomic workflows. It is shown that the contaminant profiles varied widely between microcentrifuge tubes from different manufacturers. The most suitable polypropylene tubes tested introduced 847 labware-originating contaminant m/z's when three different manufacturing batches were tested for Folch lipid extractions. Of particular concern is that 21 primary amide and fatty acid surfactants were introduced that were identical to biological endogenous lipids, 16 of which had not been previously reported as leachables from polypropylene materials. Alternatively, the use of borosilicate glassware and PTFE-lined screw caps introduced 98 different contaminant m/z's across three manufacturing batches tested for Folch extractions. Despite the overwhelming number of labware contaminants introduced, current databases and literature only facilitated the identification of 32 contaminants. To address the dearth of publicly available contaminant information, we provide a comprehensive labware contamination repository containing high-resolution m/z values, adductation information, retention times, and MS/MS spectra. This resource should prove to be valuable for researchers in detecting and distinguishing contaminants from analytes of interest. A companion paper presents a detailed study of how labware contamination can lead to ion-suppression effects on coeluting lipids and interference in the analysis of endogenous lipids, such as those from human sera.
Assuntos
Lipidômica , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Polipropilenos , Solventes/química , Ácidos GraxosRESUMO
Seasonal modifications in the structure of cellular membranes occur as an adaptive measure to withstand exposure to prolonged environmental change. Little is known about whether such changes occur independently of external cues, such as photoperiod or temperature, or how they may impact the central nervous system. We compared membrane properties of neurons isolated from the retina of goldfish (Carassius auratus), an organism well adapted to extreme environmental change, during the summer and winter months. Goldfish were maintained in a facility under constant environmental conditions throughout the year. Analysis of whole-retina phospholipid composition using mass spectrometry-based lipidomics revealed a twofold increase in phosphatidylethanolamine species during the winter, suggesting an increase in cell membrane fluidity. Atomic force microscopy was used to produce localized, nanoscale-force deformation of neuronal membranes. Measurement of Young's modulus indicated increased membrane-cortical stiffness (or decreased elasticity) in neurons isolated during the winter. Voltage-clamp electrophysiology was used to assess physiological changes in neurons between seasons. Winter neurons displayed a hyperpolarized reversal potential (Vrev) and a significantly lower input resistance (Rin) compared with summer neurons. This was indicative of a decrease in membrane excitability during the winter. Subsequent measurement of intracellular Ca2+ activity using Fura-2 microspectrofluorometry confirmed a reduction in action potential activity, including duration and action potential profile, in neurons isolated during the winter. These studies demonstrate chemical and biophysical changes that occur in retinal neurons of goldfish throughout the year without exposure to seasonal cues, and suggest a novel mechanism of seasonal regulation of retinal activity.
Assuntos
Carpa Dourada , Neurônios Retinianos , Potenciais de Ação , Animais , Carpa Dourada/fisiologia , Fotoperíodo , Estações do AnoRESUMO
Methylation of phospholipids (PL) leads to increased uniformity in positive electrospray ionization (ESI) efficiencies across the various PL subclasses. This effect is realized in the approach referred to as "trimethylation enhancement using 13C-diazomethane" (13C-TrEnDi), which results in the methyl esterification of all acidic sites and the conversion of amines to quaternary ammonium sites. Collision-induced dissociation (CID) of these cationic modified lipids enables class identification by forming distinctive headgroup fragments based on the number of 13C atoms incorporated during derivatization. However, there are no distinctive fragment ions in positive mode that provide fatty acyl information for any of the modified lipids. Gas-phase ion/ion reactions of 13C-TrEnDi-modified phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), and sphingomyelin (SM) cations with dicarboxylate anions are shown to charge-invert the positively charged phospholipids to the negative mode. An electrostatically bound complex anion is shown to fragment predominantly via a novel headgroup dication transfer to the reagent anion. Fragmentation of the resulting anionic product yields fatty acyl information, in the case of the glycerophospholipids (PE, PS, and PC), via ester bond cleavage. Analogous information is obtained from modified SM lipid anions via amide bond cleavage. Fragmentation of the anions generated from charge inversion of the 13C-TrEnDi-modified phospholipids was also found to yield lipid class information without having to perform CID in positive mode. The combination of 13C-TrEnDi modification of lipid mixtures with charge inversion to the negative-ion mode retains the advantages of uniform ionization efficiency in the positive-ion mode with the additional structural information available in the negative-ion mode without requiring the lipids to be ionized directly in both ionization modes.
Assuntos
Diazometano/química , Fosfolipídeos/química , Isótopos de Carbono/química , Estrutura Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Significant sensitivity enhancements in the tandem mass spectrometry-based analysis of complex mixtures of several phospholipid classes has been achieved via (13)C-TrEnDi. (13)C-TrEnDi-modified phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) lipids extracted from HeLa cells demonstrated greater sensitivity via precursor ion scans (PISs) than their unmodified counterparts. Sphingomyelin (SM) species exhibited neither an increased nor decreased sensitivity following modification. The use of isotopically labeled diazomethane enabled the distinction of modified PE and modified PC species that would yield isobaric species with unlabeled diazomethane. (13)C-TrEnDi created a PE-exclusive PIS of m/z 202.1, two PS-exclusive PISs of m/z 148.1 and m/z 261.1, and a PIS of m/z 199.1 for PC species (observed at odd m/z values) and SM species (observed at even m/z values). The standardized average area increase after TrEnDi modification was 10.72-fold for PE species, 2.36-fold for PC, and 1.05-fold for SM species. The sensitivity increase of PS species was not quantifiable, as there were no unmodified PS species identified prior to derivatization. (13)C-TrEnDi allowed for the identification of 4 PE and 7 PS species as well as the identification and quantitation of an additional 4 PE and 4 PS species that were below the limit of detection (LoD) prior to modification. (13)C-TrEnDi also pushed 24 PE and 6 PC lipids over the limit of quantitation (LoQ) that prior to modification were above the LoD only.
Assuntos
Diazometano/química , Fosfatidilcolinas/análise , Fosfatidiletanolaminas/análise , Fosfatidilserinas/análise , Isótopos de Carbono , Células HeLa , Humanos , Limite de Detecção , Metilação , Fosfatidilcolinas/química , Fosfatidilcolinas/classificação , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/classificação , Fosfatidilserinas/química , Fosfatidilserinas/classificação , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
A novel mass spectrometry (MS)-based lipidomics strategy that exposes glycerophospholipids to an ethereal solution of diazomethane and acid, derivatizing them to contain a net fixed, permanent positive charge, is described. The sensitivity of modified lipids to MS detection is enhanced via improved ionization characteristics as well as consolidation of ion dissociation to form one or two strong, characteristic polar headgroup fragments. Our strategy has been optimized to enable a priori prediction of ion fragmentation patterns for four subclasses of modified glycerophospholipid species. Our method enables analyte ionization regardless of proton affinity, thereby decreasing ion suppression and permitting predictable precursor ion-based quantitation with improved sensitivity in comparison to MS-based methods that are currently used on unmodified lipid precursors.
Assuntos
Diazometano/química , Espectrometria de Massas/métodos , Fosfatos/química , Glicerofosfolipídeos/química , Limite de Detecção , MetilaçãoRESUMO
The identification and quantitation of plasmalogen glycerophospholipids is challenging due to their isobaric overlap with plasmanyl ether-linked glycerophospholipids, susceptibility to acid degradation, and their typically low abundance in biological samples. Trimethylation enhancement using diazomethane (TrEnDi) can be used to significantly enhance the signal of glycerophospholipids through the creation of quaternary ammonium groups producing fixed positive charges using 13C-diazomethane in complex lipid extracts. Although TrEnDi requires a strong acid for complete methylation, we report an optimized protocol using 10 mM HBF4 with the subsequent addition of a buffer solution that prevents acidic hydrolysis of plasmalogen species and enables the benefits of TrEnDi to be realized for this class of lipids. These optimized conditions were applied to aliquots of bovine liver extract (BLE) to achieve permethylation of plasmalogen lipids within a complex mixture. Treating aliquots of unmodified and TrEnDi-derivatized BLE samples with 80% formic acid and comparing their liquid chromatography mass spectrometry (LCMS) results to analogous samples not treated with formic acid, enabled the identification of 29 plasmalogen species. On average, methylated plasmalogen species from BLE demonstrated 2.81-fold and 28.1-fold sensitivity gains over unmodified counterparts for phosphatidylcholine and phosphatidylethanolamine plasmalogen species, respectively. Furthermore, the compatibility of employing 13C-TrEnDi and a previously reported iodoacetalization strategy was demonstrated to effectively identify plasmenyl-ether lipids in complex biological extracts at greater levels of sensitivity. Overall, we detail an optimized 13C-TrEnDi derivatization strategy that enables the analysis of plasmalogen glycerophospholipids with no undesired cleavage of radyl groups, boosting their sensitivity in LCMS and LCMS/MS analyses.
Assuntos
Isótopos de Carbono , Diazometano , Glicerofosfolipídeos , Fígado , Plasmalogênios , Animais , Bovinos , Plasmalogênios/química , Plasmalogênios/análise , Isótopos de Carbono/análise , Diazometano/química , Fígado/química , Glicerofosfolipídeos/química , Glicerofosfolipídeos/análise , Metilação , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
The use of engineered viral strains such as gene therapy vectors and oncolytic viruses (OV) to selectively destroy cancer cells is poised to make a major impact in the clinic and revolutionize cancer therapy. In particular, several studies have shown that OV therapy is safe and well tolerated in humans and can infect a broad range of cancers. Yet in clinical studies OV therapy has highly variable response rates. The heterogeneous nature of tumors is widely accepted to be a major obstacle for OV therapeutics and highlights a need for strategies to improve viral replication efficacy. Here, we describe the development of a new class of small molecules for selectively enhancing OV replication in cancer tissue. Medicinal chemistry studies led to the identification of compounds that enhance multiple OVs and gene therapy vectors. Lead compounds increase OV growth up to 2000-fold in vitro and demonstrate remarkable selectivity for cancer cells over normal tissue ex vivo and in vivo. These small molecules also demonstrate enhanced stability with reduced electrophilicity and are highly tolerated in animals. This pharmacoviral approach expands the scope of OVs to include resistant tumors, further potentiating this transformative therapy. It is easily foreseeable that this approach can be applied to therapeutically enhance other attenuated viral vectors.