Rapid lipidomic profiling based on ultra-high performance liquid chromatography-mass spectrometry and its application in diabetic retinopathy.

Lipidomics aims to characterize lipid alteration in response to internal or external subtle perturbations in complex biological samples. Lipid abnormality is a major risk factor for many diseases. Large-scale lipidomic studies may offer new insights into the pathophysiological mechanisms of diseases, new opportunities in systems biology, functional biology, and personalized medicine. To this end, a highly efficient and stable lipidomic method is highly in demand. We herein present a rapid and relatively high coverage lipidomic profiling approach based on ultra-high performance liquid chromatography-mass spectrometry by comparing the performance of different chromatographic columns, optimizing the elution gradient and selecting an appropriate data acquisition mode of mass spectra. As a result, a total of 481 lipids were detected from 40 µL serum sample within 13 min, covering 20 common lipid (sub)classes. The developed method was well validated with satisfactory analytical characteristics in linearity, repeatability, stability, and lipid coverage. To show the usefulness, the method was employed to investigate serum lipid profiling of 43 subjects with mild diabetic retinopathy and 44 normal controls, and successfully defined the differential lipids related to diabetic retinopathy. We believe that this rapid method will be beneficial for lipidomic analysis of large-scale clinical samples.

A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry.

De novo fatty acid synthesis is a pivotal enzymatic process in all eukaryotic organisms. It is involved in the conversion of glucose and other nutrients to fatty acyl (FA) chains, that cells use as building blocks for membranes, energy storage, and signaling molecules. Central to this multistep enzymatic process is the cytosolic type I fatty acid synthase complex (FASN) which in mammals produces, according to biochemical textbooks, primarily non-esterified palmitic acid (NEFA 16:0). The activity of FASN is commonly measured using a spectrophotometry-based assay that monitors the consumption of the reactant NADPH. This assay is indirect, can be biased by interfering processes that use NADPH, and cannot report the NEFA chain-length produced by FASN. To circumvent these analytical caveats, we developed a simple mass spectrometry-based assay that affords monitoring of FASN activity and its product-specificity. In this assay (i) purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, (ii) at defined time points the reaction mixture is spiked with an internal NEFA standard and extracted, and (iii) the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. This assay supports essentially noise-free detection and absolute quantification of denovo synthetized 13C-labled NEFAs. We demonstrate the efficacy of our assay by determining the specific activity of purified cow FASN and show that in addition to the canonical NEFA 16:0 this enzyme also produces NEFA 12:0, 14:0, 18:0, and 20:0. We note that our assay is generic and can be carried out using commonly available high-resolution mass spectrometers with a resolving power as low as 95,000. We deem that our simple assay could be used as high-throughput screening technology for developing potent FASN inhibitors and for enzyme engineering aimed at modulating the activity and the product-landscape of fatty acid synthases.