19 research outputs found
Enhanced separations strategies for complex sample characterization using ion mobility-mass spectrometry
Protocol for mapping the metabolome and lipidome of medulloblastoma cells using liquid chromatography-mass spectrometry
Summary: Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics and lipidomics have recently been used to show that MYC-amplified group 3 medulloblastoma tumors are driven by metabolic reprogramming. Here, we present a protocol to extract metabolites and lipids from human medulloblastoma brain tumor-initiating cells and normal neural stem cells. We describe untargeted LC-MS methods that can be used to achieve extensive coverage of the polar metabolome and lipidome. Finally, we detail strategies for metabolite identification and data analysis.For complete details on the use and execution of this protocol, please refer to Gwynne et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics
Metabolic rewiring of the hypertensive kidney
Hypertension is a persistent epidemic across the developed world that is closely associated with kidney disease. Here, we applied a metabolomic, phosphoproteomic, and proteomic strategy to analyze the effect of hypertensive insults on kidneys.Our data revealed the metabolic aspects of hypertension-induced glomerular sclerosis, including lipid breakdown at early disease stages and activation of anaplerotic pathways to regenerate energy equivalents to counter stress. For example, branched-chain amino acids and proline, required for collagen synthesis, were depleted in glomeruli at early time points. Furthermore, indicators of metabolic stress were reflected by low amounts of ATP and NADH and an increased abundance of oxidized lipids derived from lipid breakdown. These processes were specific to kidney glomeruli where metabolic signaling occurred through mTOR and AMPK signaling. Quantitative phosphoproteomics combined with computational modeling suggested that these processes controlled key molecules in glomeruli and specifically podocytes, including cytoskeletal components and GTP-binding proteins, which would be expected to compete for decreasing amounts of GTP at early time points. As a result, glomeruli showed increased expression of metabolic enzymes of central carbon metabolism, amino acid degradation, and lipid oxidation, findings observed in previously published studies from other disease models and patients with glomerular damage. Overall, multilayered omics provides an overview of hypertensive kidney damage and suggests that metabolic or dietary interventions could prevent and treat glomerular disease and hypertension-induced nephropathy
Aqueous Epoxide Ring-Opening Polymerization (AEROP): Green Synthesis of Polyglycidol with Ultralow Branching
A green
synthesis method for the preparation of polyglycidol in
buffer with a high degree of control is presented. Polymerizations
were conducted in phosphate buffered saline (PBS) of varying pH 3.8,
6.0, and 8.0 and deionized water at temperatures of 60, 80, and 100
°C. Taking advantage of the catalytic reactivity between water
and glycidol afforded a novel green polymerization technique for the
facile synthesis of polyglycidol systems with semibranched architectures
with a degree of branching (DB) of 0.24, which situates the polymers
between purely linear (DB = 0) and hyperbranched (DB = 0.56–0.63)
systems. This method sidesteps the strenuous polymerization conditions
required for most polyglycidol structures and provides well-controlled
and reproducible species with a high degree of linearity and narrow
polydispersities of 1.1–1.2 via the first green and nonexothermic
synthesis of polyglycidol
Drug-Initiated Activity Metabolomics Identifies Myristoylglycine as a Potent Endogenous Metabolite for Human Brown Fat Differentiation
Worldwide, obesity rates have doubled since the 1980s and in the USA alone, almost 40% of adults are obese, which is closely associated with a myriad of metabolic diseases such as type 2 diabetes and arteriosclerosis. Obesity is derived from an imbalance between energy intake and consumption, therefore balancing energy homeostasis is an attractive target for metabolic diseases. One therapeutic approach consists of increasing the number of brown-like adipocytes in the white adipose tissue (WAT). Whereas WAT stores excess energy, brown adipose tissue (BAT) can dissipate this energy overload in the form of heat, increasing energy expenditure and thus inhibiting metabolic diseases. To facilitate BAT production a high-throughput screening approach was developed on previously known drugs using human Simpson–Golabi–Behmel Syndrome (SGBS) preadipocytes. The screening allowed us to discover that zafirlukast, an FDA-approved small molecule drug commonly used to treat asthma, was able to differentiate adipocyte precursors and white-biased adipocytes into functional brown adipocytes. However, zafirlukast is toxic to human cells at higher dosages. Drug-Initiated Activity Metabolomics (DIAM) was used to investigate zafirlukast as a BAT inducer, and the endogenous metabolite myristoylglycine was then discovered to mimic the browning properties of zafirlukast without impacting cell viability. Myristoylglycine was found to be bio-synthesized upon zafirlukast treatment and was unique in inducing brown adipocyte differentiation, raising the possibility of using endogenous metabolites and bypassing the exogenous drugs to potentially alleviate disease, in this case, obesity and other related metabolic diseases
Unusual Kinetic Isotope Effects of Deuterium Reinforced Polyunsaturated Fatty Acids in Tocopherol-Mediated Free Radical Chain Oxidations
Substitution of -CD2- at the reactive centers of linoleic and linolenic acids reduces the rate of abstraction of D by a tocopheryl radical by as much as 36-fold, compared to the abstraction of H from a corresponding -CH2- center. This H atom transfer reaction is the rate-determining step in the tocopherol-mediated peroxidation of lipids in human low-density lipoproteins, a process that has been linked to coronary artery disease. The unanticipated large kinetic isotope effects reported here for the tocopherol-mediated oxidation of linoleic and linolenic acids and esters suggests that tunneling makes this process favorable
Unusual Kinetic Isotope Effects of Deuterium Reinforced Polyunsaturated Fatty Acids in Tocopherol-Mediated Free Radical Chain Oxidations
Substitution
of −CD<sub>2</sub>– at the reactive
centers of linoleic and linolenic acids reduces the rate of abstraction
of D by a tocopheryl radical by as much as 36-fold, compared to the
abstraction of H from a corresponding −CH<sub>2</sub>–
center. This H atom transfer reaction is the rate-determining step
in the <i>tocopherol-mediated peroxidation</i> of lipids
in human low-density lipoproteins, a process that has been linked
to coronary artery disease. The unanticipated large kinetic isotope
effects reported here for the tocopherol-mediated oxidation of linoleic
and linolenic acids and esters suggests that tunneling makes this
process favorable
Data Streaming for Metabolomics: Accelerating Data Processing and Analysis from Days to Minutes
The speed and throughput
of analytical platforms has been a driving
force in recent years in the “omics” technologies and
while great strides have been accomplished in both chromatography
and mass spectrometry, data analysis times have not benefited at the
same pace. Even though personal computers have become more powerful,
data transfer times still represent a bottleneck in data processing
because of the increasingly complex data files and studies with a
greater number of samples. To meet the demand of analyzing hundreds
to thousands of samples within a given experiment, we have developed
a data streaming platform, XCMS Stream, which capitalizes on the acquisition
time to compress and stream recently acquired data files to data processing
servers, mimicking just-in-time production strategies from the manufacturing
industry. The utility of this XCMS Online-based technology is demonstrated
here in the analysis of T cell metabolism and other large-scale metabolomic
studies. A large scale example on a 1000 sample data set demonstrated
a 10 000-fold time savings, reducing data analysis time from
days to minutes. Further, XCMS Stream has the capability to increase
the efficiency of downstream biochemical dependent data acquisition
(BDDA) analysis by initiating data conversion and data processing
on subsets of data acquired, expanding its application beyond data
transfer to smart preliminary data decision-making prior to full acquisition