JLR Methods
Artifactual FA dimers mimic FAHFA signals in untargeted metabolomics pipelines
FA esters of hydroxy FAs (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Because of their low molar abundance, FAHFAs are typically quantified using highly sensitive LC-MS/MS methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point but could lead to misidentification.Development of oxaalkyne and alkyne fatty acids as novel tracers to study fatty acid beta-oxidation pathways and intermediates
Fatty acid beta-oxidation is a key process in mammalian lipid catabolism. Disturbance of this process results in severe clinical symptoms, including dysfunction of the liver, a major beta-oxidizing tissue. For a thorough understanding of this process, a comprehensive analysis of involved fatty acid and acyl-carnitine intermediates is desired, but capable methods are lacking. Here, we introduce oxaalkyne and alkyne fatty acids as novel tracers to study the beta-oxidation of long- and medium-chain fatty acids in liver lysates and primary hepatocytes.An advanced method for propargylcholine phospholipid detection by direct-infusion MS
Phospholipids with a choline head group are an abundant component of cellular membranes and are involved in many important biological functions. For studies on the cell biology and metabolism of these lipids, traceable analogues where propargylcholine replaces the choline head group have proven useful. We present a novel method to analyze propargylcholine phospholipids by MS. The routine employs 1-radyl-2-lyso-sn-glycero-3-phosphopropargylcholines as labeled lysophosphatidylcholine precursors, which upon cellular conversion direct the traceable tag with superb specificity and efficiency to the primary target lipid class.Accurate quantification of lipid species affected by isobaric overlap in Fourier-transform mass spectrometry
Lipidomics data require consideration of ions with near-identical masses, which comprises among others the Type-II isotopic overlap. This overlap occurs in series of lipid species differing only by number of double bonds (DBs) mainly because of the natural abundance of 13C-atoms. High-resolution mass spectrometry, such as Fourier-transform mass spectrometry (FTMS), is capable of resolving Type-II overlap depending on mass resolving power. In this work, we evaluated FTMS quantification accuracy of lipid species affected by Type-II overlap.A liquid chromatography-mass spectrometry workflow for in-depth quantitation of fatty acid double bond location isomers
Tracing compositional changes of fatty acids (FAs) is frequently used as a means of monitoring metabolic alterations in perturbed biological states. Given that more than half of FAs in the mammalian lipidome are unsaturated, quantitation of FAs at a carbon-carbon double bond (C=C) location level is necessary. The use of 2-acetylpiridine (2-acpy) as the charge-tagging PB reagent led to a limit of identification in the subnanomolar range for mono- and polyunsaturated as well as conjugated FAs. Conjugated free FAs of low abundance such as FA 18:2 (n-7, n-9) and FA 18:2 (n-6, n-8) were quantified at concentrations of 0.61 ± 0.05 and 0.05 ± 0.01 mg per 100 g in yak milk powder, respectively.
