A parallel reaction monitoring-mass spectrometric method for studying lipid biosynthesis in vitro using 13C16-palmitate as an isotope tracer

Abstract

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.

Keywords: HILIC-MS/MS; Isotope tracing; Lipid biosynthesis; Lipidomics; Parallel reaction monitoring.