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.