The TripleTOF 5600 System, a hybrid triple quadrupole time-of-flight mass spectrometer, was evaluated to explore the key figures of merit in generating peptide and protein identifications which included spectral acquisition rates, data quality, proteome coverage, and biological depth. Employing a Saccharomyces cerevisiae tryptic digest, careful consideration of several performance features demonstrated that the speed of the TripleTOF contributed most to the resultant data. The TripleTOF system was operated with 8, 20, and 50 MS/MS events in an effort to compare to other MS technologies and to demonstrate the abilities of the instrument platform.
Non-alcoholic fatty liver disease (NAFLD) is associated with altered hepatic lipid composition.Animal studies suggest that the hepatic ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) contributes to steatogenesis and inflammation. This ratio may be influenced by dysregulation of PE N-methyltransferase (PEMT) pathway or low choline diet.Alterations in the liver may also influence lipid composition in circulation such as in erythrocytes, which therefore may have utility as a biomarker of hepatic disease. Currently, no study has assessed both liver and erythrocyte PC/PE ratios in NAFLD. Aim was to compare PC/PE ratio in liver and erythrocytes of patients with simple steatosis (SS) or steatohepatitis (NASH) to healthy controls. PC and PE were measured by mass spectrometry in 28 patients with biopsy proven NAFLD (14 SS, 14 NASH) and 9 healthy living liver donors as controls. The hepatic PC/PE ratio was lower in SS (median [range]) (1.23 [0.27-3.40]) and ]) compared to controls (3.14 [2.20-3.73]); both P<0.001), but it was not different between SS and NASH. PC was lower and PE higher in the liver of SS patients compared to controls, whereas in NASH only PE was higher. The PC/PE ratio in erythrocytes was also lower in SS and NASH compared to controls, due to lower PC in both patient groups. PE in erythrocytes was not different among the groups. In conclusion, NAFLD patients have lower PC/PE ratio in liver and erythrocytes than healthy controls, which may play a role in the pathogenesis. Underlying mechanisms require further investigation.
Shotgun lipidomics has evolved into a myriad of multi-dimensional strategies for molecular lipid characterization, including bioinformatics tools for mass spectrum interpretation and quantitative measurements to study systems-lipidomics in complex biological extracts. Taking advantage of spectral mass accuracy, scan speed and sensitivity of improved quadrupole linked time-of-flight mass analyzers, we developed a bias-free global lipid profiling acquisition technique of sequential precursor ion fragmentation called MS/MSALL. This generic information-independent tandem mass spectrometry (MS) technique consists of a Q1 stepped mass isolation window through a set mass range in small increments, fragmenting and recording all product ions and neutral losses. Through the accurate MS and MS/MS information, the molecular lipid species are resolved, including distinction of isobaric and isomeric species, and composed into more precise lipidomic outputs. The method demonstrates good reproducibility and at least 3 orders of dynamic quantification range for isomeric ceramides in human plasma. More than 400 molecular lipids in human plasma were uncovered and quantified in less than 12 min, including acquisitions in both positive and negative polarity modes. We anticipate that the performance of sequential precursor ion fragmentation both in quality and throughput will lead to the uncovering of new avenues throughout the biomedical research community, enhance biomarker discovery and provide novel information target discovery programs as it will prospectively shed new insight into affected metabolic and signaling pathways.
High NaCl elevates activity of the osmoprotective transcription factor TonEBP/OREBP by increasing its phosphorylation, transactivating activity, and localization to the nucleus. We investigated the possible role in this activation of phospholipase C-γ1 (PLC-γ1), which has a predicted binding site at TonEBP/OREBPphospho-Y143. We find the following. The PLC-γ1 phospholipase inhibitor U72133 inhibits nuclear localization of TonEBP/OREBP but not the increase of its transactivating activity. We conclude that, when NaCl is elevated, TonEBP/OREBP becomes phosphorylated at Y143, resulting in binding of PLC-γ1 to that site, which contributes to TonEBP/OREBP transcriptional activity, transactivating activity, and nuclear localization.hypertonicity | phosphorylation | proteomics
This article is available online at http://www.jlr.org Acyl-CoAs are a class of important molecules that play essential roles in many physiological processes ( 1 ), such as fatty acid oxidation, lipid synthesis/remodeling, ketone body synthesis, xenobiotic metabolism, and signaling pathways. Classically, acyl-CoAs such as free CoA, acetyl-CoA, and malonyl-CoA are recognized as regulators of metabolic fl ux. The ratio of acetyl-CoA versus free CoA tightly regulates glycolysis and fatty acid oxidation. Malonyl-CoA attenuates fatty acid oxidation by inhibiting acyl-CoA transport into the mitochondrion for oxidation and is utilized in fatty acid synthesis when its concentration is elevated ( 2 ). Many proteins and genes are dynamically regulated by deacylation and acylation via various acyl-CoAs, such as acetyl-CoA, succinyl-CoA, palmitoyl-CoA, etc. ( 3 ). However, acyl-CoAs present in the cell are diverse and may involve molecules beyond fatty acids and their oxidized derivatives. The metabolism of xenobiotics can also lead to the formation of acyl-CoAs, as demonstrated in our previous work on the metabolism of 4-hydroxy acids from C 4 to C 11 in perfused rat liver. The identifi cation of these novel acyl-CoAs extends our understanding of the new catabolic pathways involved in the disposal of 4-hydroxy acids including drugs of abuse and lipid peroxidation products ( 4-8 ).Acyl-CoAs are exclusive to the intracellular metabolites, and the profi le of these biomolecules is indicative of the local metabolic status. Each organ has its specifi c physiological roles with different energetic demands, and therefore, would be expected to exhibit a unique acyl-CoA profi le. The heart preferentially utilizes fatty acids to meet the ATP demand of mechanical contraction. Glucose and/or ketone bodies serve as the primary substrate of the brain for energy
Aims/hypothesis To directly assess the role of beta cell lipolysis in insulin secretion and whole-body energy homeostasis, inducible beta cell-specific adipose triglyceride lipase (ATGL)-deficient (B-Atgl-KO) mice were studied under normal diet (ND) and high-fat diet (HFD) conditions. Methods Atgl flox/flox mice were cross-bred with Mip-Cre-ERT mice to generate Mip-Cre-ERT /+ ;Atgl flox/flox mice. At 8 weeks of age, these mice were injected with tamoxifen to induce deletion of beta cell-specific Atgl (also known as Pnpla2), and the mice were fed an ND or HFD.Results ND-fed male B-Atgl-KO mice showed decreased insulinaemia and glucose-induced insulin secretion (GSIS) in vivo. Changes in GSIS correlated with the islet content of long-chain saturated monoacylglycerol (MAG) species that have been proposed to be metabolic coupling factors for insulin secretion. Exogenous MAGs restored GSIS in B-Atgl-KO islets. B-Atgl-KO male mice fed an HFD showed reduced insulinaemia, glycaemia in the fasted and fed states and after glucose challenge, as well as enhanced insulin sensitivity. Moreover, decreased insulinaemia in B-Atgl-KO mice was associated with increased energy expenditure, and lipid metabolism in brown (BAT) and white (WAT) adipose tissues, leading to reduced fat mass and body weight. Conclusions/interpretation ATGL in beta cells regulates insulin secretion via the production of signalling MAGs. Decreased insulinaemia due to lowered GSIS protects B-Atgl-KO mice from diet-induced obesity, improves insulin sensitivity, increases lipid mobilisation from WAT and causes BAT activation. The results support the concept that fuel excess can drive obesity and diabetes via hyperinsulinaemia, and that an islet beta cell ATGL-lipolysis/adipose tissue axis controls energy homeostasis and body weight via insulin secretion.
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