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Updated: May 10, 2025
Shotgun Lipidomics of Rodent Tissues
Published on: November 18, 2022
A parallel reaction monitoring-mass spectrometric method for studying lipid biosynthesis in vitro using
Kyeong-Seog Kim1, Young Gyun Ko2, Woo Seok Yang2
1Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 03080, Republic of Korea; Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, Seoul, 03080, Republic of Korea.
Background:
Palmitate, which is the end product of fatty acid synthase, is the key fatty acid for understanding of lipid biosynthetic process in mammalian cells. Mass spectrometry (MS) methodology using 13C-palmitate can trace the lipid biosynthesis such as glycerolipids, glycerophospholipids, and sphingolipids. However, due to the interferences of natural heavy isotopes, accurate measurement of 13C-labeled lipid species has been limited. Here we describe a high-throughput isotope tracing experiment to assess lipid biosynthesis using parallel reaction monitoring-MS (PRM-MS) with 13C16-palmitate as an isotope tracer.
Results:
The developed method can trace 14 13C16-labeled lipid classes without disturbance from the heavy isotope patterns of natural lipids. Lipid class-based separation was achieved through hydrophilic interaction liquid chromatography (HILIC) which allows facile identification of lipid, and PRM-MS was performed for accurate detection of the 13C16-labeled lipids. A fibroblast (NIH/3T3) cell line was used as an in vitro model, and the NIH/3T3 cells were treated with bovine serum albumin (BSA)-bound 13C16-palmitate. The isotopic disturbance from natural lipid was eliminated using 13C16-palmitate, rather than 13C1-palmitate, as an isotope tracer. After 24 h of incubation with 0.1 mmol/L of BSA-bound 13C16-palmitate in the fibroblasts, NIH/3T3 cells synthesized the 127 13C16-labeled lipid species of glycerolipids, glycerophospholipids, and sphingolipids. Finally, in the NIH/3T3 cells incubated for 1, 6, and 24 h after the treatment of the isotope tracer exhibited an increased profile of 13C16-labeled lipidome, depending on duration of incubation.
Significance:
The HILIC/PRM-MS method using 13C16-palmitate as an isotope tracer enables identification of 13C16-labeled lipid species by annotating 13C16-labeled position, including the 13C16-fatty acyl chain and 13C16-sphingolipid headgroup, without interference of natural heavy isotope patterns. This lipidomic flux analysis using PRM approach is expected to provide insights into assessment of isotope-labeled lipids.
