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Metabolic Heavy Isotope Labeling to Study Glycerophospholipid Homeostasis of Cultured Cells.

Bio-protocol (2017-05-05)
Satu Hänninen, Pentti Somerharju, Martin Hermansson
ABSTRACT

Glycerophospholipids consist of a glycerophosphate backbone to which are esterified two acyl chains and a polar head group. The head group (e.g., choline, ethanolamine, serine or inositol) defines the glycerophospholipid class, while the acyl chains together with the head group define the glycerophospholipid molecular species. Stable heavy isotope (e.g., deuterium)-labeled head group precursors added to the culture medium incorporate efficiently into glycerophospholipids of mammalian cells, which allows one to determine the rates of synthesis, acyl chain remodeling or turnover of the individual glycerophospholipids using mass spectrometry. This protocol describes how to study the metabolism of the major mammalian glycerophospholipids i.e., phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and phosphatidylinositols with this approach.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hydroxylamine hydrochloride, puriss. p.a., ACS reagent, ≥99.0% (RT)
Sigma-Aldrich
myo-Inositol, ≥99%
Supelco
Ammonium formate, eluent additive for LC-MS, LiChropur, ≥99.0%
Sigma-Aldrich
L-Serine, ReagentPlus®, ≥99% (HPLC)
Sigma-Aldrich
Choline chloride, ≥98%
Sigma-Aldrich
Fetal Bovine Serum, non-USA origin, sterile-filtered, suitable for cell culture