Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry.
Triacylglycerols (TAGs) are neutral lipids present in all mammalian cells as energy reserves, and diacylglycerols (DAGs) are present as intermediates in phospholipid biosynthesis and as signaling molecules. The molecular species of TAGs and DAGs present in mammalian cells are quite complex, and previous investigations revealed multiple isobaric species having molecular weights at virtually every even mass between 600 and 900 Da, making it difficult to assess changes of individual molecular species after cell activation. A method has been developed, using tandem MS and neutral loss scanning, to quantitatively analyze changes in those glyceryl ester molecular species containing identical fatty acyl groups. This was carried out by neutral loss scanning of 18 common fatty acyl groups where the neutral loss corresponded to the free carboxylic acid plus NH(3). Deuterium-labeled internal standards were used to normalize the signal for each nominal [M+NH(4)](+) ion undergoing this neutral loss reaction. This method was applied in studies of TAGs in RAW 264.7 cells treated with the toll-like receptor 4 ligand Kdo(2)-lipid A. A 50:1-TAG containing 18:1 was found to increase significantly over a 24-h time course after Kdo(2)-lipid A exposure, whereas an isobaric 50:1-TAG containing 16:1 did not change relative to controls.
Murphy
Robert C
RC
Department of Pharmacology, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA. robert.murphy@uchsc.edu
James
Patrick F
PF
McAnoy
Andrew M
AM
Krank
Jessica
J
Duchoslav
Eva
E
Barkley
Robert M
RM
D000818:Animals; D002460:Cell Line; D003903:Deuterium; D004075:Diglycerides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D015394:Molecular Structure; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards; D051197:Toll-Like Receptor 4 / Q000187:drug effects / Q000378:metabolism; D014280:Triglycerides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D004075:Diglycerides; C506188:Kdo2-lipid A; D008024:Ligands; D008070:Lipopolysaccharides; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014280:Triglycerides; D003903:Deuterium