11 research outputs found
Comparison of in vivo solid-phase microextraction (SPME) to Folch extraction of brain lipids in Rattus norvegicus using LC-MS
Lipidomics is the comprehensive study of the lipids present in an organism. To achieve high analytical sensitivity and good lipid coverage in global lipidomics, sample preparation, mobile phase composition, chromatographic separation, and data processing parameters must be optimized. Solid-phase microextraction (SPME) is a non-exhaustive, in vivo sample preparation method that has not previously been applied to lipidomics studies but that provides a promising alternative to microdialysis for in vivo lipid analysis. Before SPME can be successfully applied to lipidomic studies, it is important to increase the sensitivity of liquid chromatography-mass spectrometry (LC-MS) detection as much as possible. This was accomplished by comparing the effect of four different mobile phase additives on signal intensity using negative electrospray ionization (ESI): acetic acid, ammonium acetate, ammonium hydroxide and ammonium acetate with acetic acid. Acetic acid at a concentration of 3.5 mM outperformed the other additives tested causing a 42% increase in lipid coverage along with a 2-19-fold increase in signal compared to 10 mM ammonium acetate. The main disadvantages of acetic acid were (i) wide peak shape for two lipid classes: phosphatidic acid and phosphatidylserine and (ii) reduction of signal intensity for phosphatidylcholine and ceramide lipids. These results show that although basic pH promotes in-solution ionization, surface, electrochemical and gas-phase processes provide significant contributions to overall ESI efficiency thus resulting in enhanced ionization at acidic pH. Acetic acid is good choice of additive for negative mode ESI due its ability to increase formation of deprotonated adducts, low molecular volume, high gas phase proton affinity, and capability for electrochemical reduction. Traditionally, lipids are extracted from brain tissue using the Folch method, a biphasic liquid-liquid extraction method that relies on chloroform/methanol solvent. In this study, SPME was compared to the Folch method to determine if it is a viable alternative. The main SPME parameters that affect extraction efficiency of in vivo SPME were evaluated for this application: extraction time, desorption time, and desorption solvent. Analysis of SPME extracts showed 147 and 613 features versus 1368 and 1161 detected in the Folch extract in positive mode and negative mode, respectively. Using LipidSearch 127 lipids in positive mode and 57 lipids in negative mode were identified in the SPME extracts verses 145 in positive mode and 99 in negative mode for the Folch extracts. This study represents the first step towards the development and inter-laboratory validation of in vivo SPME demonstrating its ability as a new and viable alternative to Folch extractions that can be used in lipidomics studies of the brains of alive, awake animals for the first time
Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS
The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34–80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics
Mitochondrial hyperfusion via metabolic sensing of regulatory amino acids
The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate
Assessment of solid phase microextraction as a sample preparation tool for untargeted analysis of brain tissue using liquid chromatography-mass spectrometry
This work presents an evaluation of solid-phase microextraction (SPME) SPME in combination with liquid chromatography-high resolution mass spectrometry (LC-HRMS) as an analytical approach for untargeted brain analysis. The study included a characterization of the metabolite coverage provided by C18, mixed-mode (MM, with benzene sulfonic acid and C18 functionalities), and hydrophilic lipophilic balanced (HLB) particles as sorbents in SPME coatings after extraction from cow brain homogenate at static conditions. The effects of desorption solvent, extraction time, and chromatographic modes on the metabolite features detected were investigated. Method precision and absolute matrix effects were also assessed. Among the main findings of this work, it was observed that all three tested coating chemistries were able to provide comparable brain tissue information. HLB provided higher responses for polar metabolites; however, as these fibers were prepared in-house, higher inter-fiber relative standard deviations were also observed. C18 and HLB coatings offered similar responses with respect to lipid-related features, whereas MM and C18 provided the best results in terms of method precision. Our results also showed that the use of methanol is essential for effective desorption of non-polar metabolites. Using a reversed-phase chromatographic method, an average of 800 and 1200 brain metabolite features detected in positive and negative modes, respectively, met inter-fibre RSD values below 30% (n=4) after removal of fibre and solvent artefacts from the associated datasets. For features detected using a lipidomics method, a total of 900 and 1800 features detected using C18 fibers in positive and negative mode, respectively, met the same criteria. In terms of absolute matrix effects, the majority of the model metabolites tested showed values between 80 and 120%, which are within the acceptable range. Overall, the findings of this work lay the foundation for further optimization of parameters for SPME-LC-HRMS methods suitable for in vivo and ex vivo brain (and other tissue) untargeted studies, and support the applicability of this approach for non-destructive tissue metabolomics
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Methionine oxidation activates pyruvate kinase M2 to promote pancreatic cancer metastasis.
Cancer mortality is primarily a consequence of its metastatic spread. Here, we report that methionine sulfoxide reductase A (MSRA), which can reduce oxidized methionine residues, acts as a suppressor of pancreatic ductal adenocarcinoma (PDA) metastasis. MSRA expression is decreased in the metastatic tumors of PDA patients, whereas MSRA loss in primary PDA cells promotes migration and invasion. Chemoproteomic profiling of pancreatic organoids revealed that MSRA loss results in the selective oxidation of a methionine residue (M239) in pyruvate kinase M2 (PKM2). Moreover, M239 oxidation sustains PKM2 in an active tetrameric state to promote respiration, migration, and metastasis, whereas pharmacological activation of PKM2 increases cell migration and metastasis in vivo. These results demonstrate that methionine residues can act as reversible redox switches governing distinct signaling outcomes and that the MSRA-PKM2 axis serves as a regulatory nexus between redox biology and cancer metabolism to control tumor metastasis
Mitochondrial hyperfusion via metabolic sensing of regulatory amino acids
The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate
Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950–Metabolites in Frozen Human Plasma
As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvemen
Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma
10.1194/jlr.M079012JOURNAL OF LIPID RESEARCH58122275-228
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Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.
As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement
Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma
As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra-and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium.jlr While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement