An in silico MS/MS library for automatic annotation of novel FAHFA lipids.

BackgroundA new lipid class named 'fatty acid esters of hydroxyl fatty acids' (FAHFA) was recently discovered in mammalian adipose tissue and in blood plasma and some FAHFAs were found to be associated with type 2 diabetes. To facilitate the automatic annotation of FAHFAs in biological specimens, a tandem mass spectra (MS/MS) library is needed. Due to the limitation of the commercial available standard compounds, we proposed building an in silico MS/MS library to extend the coverage of molecules.ResultsWe developed a computer-generated library with 3267 tandem mass spectra (MS/MS) for 1089 FAHFA species. FAHFA spectra were generated based on authentic standards with negative mode electrospray ionization and 10, 20, and 40 V collision induced dissociation at 4 spectra/s as used in in ultra-high performance liquid chromatography-QTOF mass spectrometry studies. However, positional information of the hydroxyl group is only obtained either at lower QTOF spectra acquisition rates of 1 spectrum/s or at the MS(3) level in ion trap instruments. Therefore, an additional set of 4290 fragment-rich MS/MS spectra was created to enable distinguishing positional FAHFA isomers. The library was generated based on ion fragmentations and ion intensities of FAHFA external reference standards, developing a heuristic model for fragmentation rules and extending these rules to large swaths of computer-generated structures of FAHFAs with varying chain lengths, degrees of unsaturation and hydroxyl group positions. Subsequently, we validated the new in silico library by discovering several new FAHFA species in egg yolk, showing that this library enables high-throughput screening of FAHFA lipids in various biological matrices.ConclusionsThe developed library and templates are freely available for commercial or noncommercial use at http://fiehnlab.ucdavis.edu/staff/yanma/fahfa-lipid-library. This in silico MS/MS library allows users to annotate FAHFAs from accurate mass tandem mass spectra in an easy and fast manner with NIST MS Search or PepSearch software. The developing template is provided for advanced users to modify the parameters and export customized libraries according to their instrument features. Graphical abstractExample of experimental and in silico MS/MS spectra for FAHFA lipids

The production of FAHFA is enhanced when Haematococcus pluvialis is grown in CO2

Microalgae are considered as a potential source of bioactive compounds to be used in different fields including food and pharmaceutical industry. In this context, fatty acid esters of hydroxy-fatty acids (FAHFA) are emerging as a new class of compounds with anti-inflammatory and anti-diabetic properties. An existing gap in the field of algal research is the limited knowledge regarding the production of these compounds. Our research questions aimed to determine whether the microalga H. pluvialis can synthesize FAHFA and whether the production levels of these compounds are increased when cultivated in a CO2-rich environment. To answer these questions, we used a LC-QTOF/MS method for the characterization of FAHFA produced by H. pluvialis while an LC-MS/MS method was used for their quantitation. The cultivation conditions of H. pluvialis, which include the utilization of CO2, can result in a 10–50-fold increase in FAHFA production

Palmitic Acid Hydroxystearic Acids Activate GPR40, Which Is Involved in Their Beneficial Effects on Glucose Homeostasis.

Palmitic acid hydroxystearic acids (PAHSAs) are endogenous lipids with anti-diabetic and anti-inflammatory effects. PAHSA levels are reduced in serum and adipose tissue of insulin-resistant people and high-fat diet (HFD)-fed mice. Here, we investigated whether chronic PAHSA treatment enhances insulin sensitivity and which receptors mediate PAHSA effects. Chronic PAHSA administration in chow- and HFD-fed mice raises serum and tissue PAHSA levels ∼1.4- to 3-fold. This improves insulin sensitivity and glucose tolerance without altering body weight. PAHSA administration in chow-fed, but not HFD-fed, mice augments insulin and glucagon-like peptide (GLP-1) secretion. PAHSAs are selective agonists for GPR40, increasing Ca+2 flux, but not intracellular cyclic AMP. Blocking GPR40 reverses improvements in glucose tolerance and insulin sensitivity in PAHSA-treated chow- and HFD-fed mice and directly inhibits PAHSA augmentation of glucose-stimulated insulin secretion in human islets. In contrast, GLP-1 receptor blockade in PAHSA-treated chow-fed mice reduces PAHSA effects on glucose tolerance, but not on insulin sensitivity. Thus, PAHSAs activate GPR40, which is involved in their beneficial metabolic effects.Supported by NIH grants R01 DK43051, P30 DK57521 (B.B.K.), and R01 DK106210 (B.B.K. and A. Saghatelian); a grant from the JPB Foundation (B.B.K.); and T32DK07516 (B.B.K. and J.L.)

Comprehensive identification of sphingolipid species by in silico retention time and tandem mass spectral library

Additional file 1. Figure S1 The fragment assignments of 12 sphingolipid classes. The annotations were combinatorially performed by hydrogen rearrangement rules in combination with substantial manual curation. The original spectra were obtained from LC/MS data of some biological samples including human cells, mouse tissues, and plant species

Targeted analysis of branched-chain faty acid esters by LC/MS

Lipidomika je jedna z oblastí metabolomiky, která se v poslední době, díky rozvoji analytických metod, těší velké pozornosti. Tato práce se věnuje optimalizaci extrakce lipidů z biologických vzorků, pre-separaci pomocí extrakce kapalina kapalina, extrakci na pevné fázi, následné separaci a identifikaci větvených esterů hydroxylovaných mastných kyselin (FAHFA - branched-chain Fatty Acid esters of Hydroxy Fatty Acids) pomocí ultra účinné kapalinové chromatografie spojené s hmotnostní spektrometrií. Tato třída lipidových molekul souvisí se sekrecí insulinu, schopností působit na metabolismus glukosy a poskytuje potenciál v léčbě diabetu druhého typu. Extrakce na pevné fázi byla testována na kolonkách reverzní fáze obsahujících sorbent modifikovaný styren divinylbenzenovým polymerem nebo polymerem s vázaným oktadecylem a silikagelových kolonkách pro normální fázi. Zvolená kolonka Strata SI-1 Silica byla použitapro separaci FAHFA z komplexních biologických vzorků. FAHFA analýza byla optimalizována na sestavě UPLC Ultimate 3000 RSLC vybavené chromatografickou kolonou Kinetex C18 1,7 µm, 2,1 x 150 mm v gradientovém programu. UPLC byla spojena s hmotnostním spektrometrem QTRAP 5500/SelexION pracujícím v nastavení hybridní trojitý kvadrupól/lineární iontová past a přidanou iontovou mobilitní celou....Lipidomics as a part of metabolomics is a fast-growing area of research due to the improvement in analytical techniques. This master thesis is focused on lipid extraction techniques optimization, using liquid liquid extraction and solid phase extraction as pre-separation methods and ultra performance liquid chromatography coupled with mass spectrometry for extraction and subsequent identification of branched-chain fatty acid esters (FAHFA - branched-chain Fatty Acid esters of Hydroxy Fatty Acids). This newly discovered class of lipid molecules is associated with insulin secretion, which could improve whole body and local glucose metabolism, providing potential for diabetes 2 type treatment. Solid phase extraction of biological samples was optimized on columns regarding to sorbent composition using reversed phase columns with modified styrene divinylbenzene polymer or octadecyl-bonded polymer and normal phase columns packed with silica gel. Column Strata SI-1 Silica was the most effective for FAHFA separation from biological samples. Chromatographic separation of FAHFA was performed on UPLC Ultimate 3000 RSLC equipped with Kinetex C18 1,7 µm, 2,1 x 150 mm column using gradient program. UPLC was coupled to QTRAP 5500/SelexION, a hybrid, triple quadrupole, linear ion trap mass spectrometer equipped...Katedra analytické chemieDepartment of Analytical ChemistryPřírodovědecká fakultaFaculty of Scienc

An in silico MS/MS library for automatic annotation of novel FAHFA lipids

BACKGROUND: A new lipid class named ‘fatty acid esters of hydroxyl fatty acids’ (FAHFA) was recently discovered in mammalian adipose tissue and in blood plasma and some FAHFAs were found to be associated with type 2 diabetes. To facilitate the automatic annotation of FAHFAs in biological specimens, a tandem mass spectra (MS/MS) library is needed. Due to the limitation of the commercial available standard compounds, we proposed building an in silico MS/MS library to extend the coverage of molecules. RESULTS: We developed a computer-generated library with 3267 tandem mass spectra (MS/MS) for 1089 FAHFA species. FAHFA spectra were generated based on authentic standards with negative mode electrospray ionization and 10, 20, and 40 V collision induced dissociation at 4 spectra/s as used in in ultra-high performance liquid chromatography-QTOF mass spectrometry studies. However, positional information of the hydroxyl group is only obtained either at lower QTOF spectra acquisition rates of 1 spectrum/s or at the MS(3) level in ion trap instruments. Therefore, an additional set of 4290 fragment-rich MS/MS spectra was created to enable distinguishing positional FAHFA isomers. The library was generated based on ion fragmentations and ion intensities of FAHFA external reference standards, developing a heuristic model for fragmentation rules and extending these rules to large swaths of computer-generated structures of FAHFAs with varying chain lengths, degrees of unsaturation and hydroxyl group positions. Subsequently, we validated the new in silico library by discovering several new FAHFA species in egg yolk, showing that this library enables high-throughput screening of FAHFA lipids in various biological matrices. CONCLUSIONS: The developed library and templates are freely available for commercial or noncommercial use at http://fiehnlab.ucdavis.edu/staff/yanma/fahfa-lipid-library. This in silico MS/MS library allows users to annotate FAHFAs from accurate mass tandem mass spectra in an easy and fast manner with NIST MS Search or PepSearch software. The developing template is provided for advanced users to modify the parameters and export customized libraries according to their instrument features. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13321-015-0104-4) contains supplementary material, which is available to authorized users

Synthesis, SAR library, and chemical biology probes of PAHSAs, a family of natural product lipids with anti-diabetic activity

Nearly one in ten Americans has type 2 diabetes mellitus (T2DM), a major source of morbidity, mortality, and healthcare costs. Most patients with diabetes are treated with medicines which may have adverse side effects. The prevalence of this disease necessitates the addition of new, safer compounds to the anti-diabetic arsenal. The recently reported Fatty Acid esters of Hydroxy Fatty Acids (FAHFAs) included eight Palmitic Acid esters of Hydroxy Stearic Acid (PAHSA) regioisomers, which may provide a new avenue for treatment of T2DM. In particular, 5- and 9-PAHSA increased insulin secretion, improved blood sugar, and decreased adipose tissue inflammation in diabetic mice. While this activity is encouraging, much remains unknown about PAHSAs, including the influence of chemical connectivity on their activity. Furthermore, the molecular targets of PAHSAs have not been fully elucidated. Until these questions are answered, there can be no hope of treating T2DM with an optimized PAHSA analog, nor means to rationally upregulate de novo PAHSA synthesis. This resulted in a scalable total synthesis of racemic 5- and 9-PAHSA, both enantiomers of 9-PAHSA, and preparation of a 30 membered library of PAHSA analogs to probe structure activity relationships. Furthermore, 6 chemical biology probes have been prepared to determine in an unbiased manner the molecular targets of PAHSAs and their metabolism.Chemistr

MOESM3 of An in silico MS/MS library for automatic annotation of novel FAHFA lipids

Additional file 3. Annotation results of the METLIN online reference spectra by the in silico library

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Additional file 2. Common fatty acids included in the FAHFA in silico library