In vitro and in situ tracking of choline-phospholipid biogenesis by MALDI TOF-MS.
The quantitative monitoring of newly synthesized species of phosphatidylcholines (PCs) and sphingomyelins (SMs) has been achieved in cultured human lens epithelial cells, both in situ and in vitro, with the use of MALDI TOF-MS. As the cells were cultured with deuterated choline-d(9), new peaks that differed from the hydrogenated species by 9.06 Da appeared in the mass spectra. The initial rates of appearance of all deuterated species of PCs were comparable and 4 times higher than those for SMs. After 12 h, those rates began to decrease for PCs but not for deuterated SMs, whose relative contents continued to increase throughout the 72 h of the experiment. The differences in initial rates are consistent with the reported initial generation of PCs, their subsequent degradation, and transfer of their headgroup, phosphorylcholine, to SMs. To further test the ability of MALDI TOF-MS to quantify changes in phospholipid (PL) metabolic pathways, myriocin, an inhibitor of SM synthesis, was added to the cells. In vitro and in situ results revealed a decrease in SMs and an unexpected increase in some PCs. With the use of other deuterated precursors and in combination with postsource decay or tandem MS/MS, this approach could allow the simultaneous tracking of the biosynthesis of multiple PL classes while providing details on their acyl chains.
Department of Chemistry, University of Louisville, Louisville, KY 40292, USA.
Rosendo
R
Estrada
Douglas
D
Borchman
John
J
Reddan
Anne
A
Hitt
M Cecilia
MC
Yappert
D002460:Cell Line; D010767:Phosphorylcholine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010767:Phosphorylcholine