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.A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound
MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens.Analytical separations for lipids in complex, nonpolar lipidomes using differential mobility spectrometry
Secretions from meibomian glands located within the eyelid (commonly known as meibum) are rich in nonpolar lipid classes incorporating very-long (22–30 carbons) and ultra-long (>30 carbons) acyl chains. The complex nature of the meibum lipidome and its preponderance of neutral, nonpolar lipid classes presents an analytical challenge, with typically poor chromatographic resolution, even between different lipid classes. To address this challenge, we have deployed differential mobility spectrometry (DMS)-MS to interrogate the human meibum lipidome and demonstrate near-baseline resolution of the two major nonpolar classes contained therein, namely wax esters and cholesteryl esters.DMS as an orthogonal separation to LC/ESI/MS/MS for quantifying isomeric cerebrosides in plasma and cerebrospinal fluid
Cerebrosides, including glucosylceramides (GlcCers) and galactosylceramides (GalCers), are important membrane components of animal cells with deficiencies resulting in devastating lysosomal storage disorders. Their quantification is essential for disease diagnosis and a better understanding of disease mechanisms. The simultaneous quantification of GlcCer and GalCer isomers is, however, particularly challenging due to their virtually identical structures. To address this challenge, we developed a new LC/MS-based method using differential ion mobility spectrometry (DMS) capable of rapidly and reproducibly separating and quantifying isomeric cerebrosides in a single run.Mass spectrometry imaging of lipids: untargeted consensus spectra reveal spatial distributions in Niemann-Pick disease type C1
Mass spectrometry imaging (MSI) is a tool to rapidly map the spatial location of analytes without the need for tagging or a reporter system. Niemann-Pick disease type C1 (NPC1) is a neurodegenerative, lysosomal storage disorder characterized by accumulation of unesterified cholesterol and sphingolipids in the endo-lysosomal system. Here, we use MSI to visualize lipids including cholesterol in cerebellar brain tissue from the NPC1 symptomatic mouse model and unaffected controls. To complement the imaging studies, a data-processing pipeline was developed to generate consensus mass spectra, thereby using both technical and biological image replicates to assess differences.
