Mass spectrometry determination of endonuclear phospholipid composition and dynamics

Mammalian cell lipid analyses using tandem electrospray ionization mass spectrometry, in conjunction with stable isotope labeling, permit unparalleled access to membrane phospholipid molecular species compositions and turnover. Lipidomic data from isolable compartments of lipid second messenger generation, such as membrane-free nuclei, can provide dynamic insights into the topology of phospholipid turnover. For example, ESI-MS/MS precursor scans of characteristic phosphocholine m/z 184+ fragments reveal a highly saturated endonuclear phosphatidylcholine pool with homeostatic maintenance properties. A spatially distinct CDPcholine pathway yields, within minutes of choline-d9 labeling, unsaturated endonuclear phosphatidylcholines progressively remodeled to more saturated species evidenced by tracking the deuteriated headgroup through precursor scans of phosphocholine-d9 (m/z 193+ fragment). Among the other endonuclear phospholipids, diacyl phosphatidylethanolamines (neutral loss of m/z 141+) are also highly saturated compared with those of whole cell whereas, phophatidylinositols (precursor scans of m/z 241− fragment) are essentially identical in nuclei and whole cells. Moreover, the pattern of myo-inositol-d6 acquisition into endonuclear phosphatidylinositol (precursor scans of m/z 247− fragment) is inconsistent with compartment-specific synthesis. Endonuclear sphingomyelins (seen in precursor scans of m/z 184+ and confirmed from precursor scans of m/z 168− fragments) are enriched but similar in composition to whole cell species whereas endonuclear phosphatidylserines (neutral loss of m/z 87−) are more saturated than their whole cell counterparts. The focus of described methodologies emphasize their value in probing the compositions and dynamics of endonuclear phospholipids, but in principle may be extended to exploration of other isolable compartments including ER or plasma membranes.