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Abstract:

Cardiolipins (CL) are complex mitochondrial-specific phospholipids that contribute to the regulation of multiple mitochondrial signaling functions and bioenergetics in mammalian cells. Here, we present a method to quantify and characterize CL molecular species from lipid extracts of isolated mitochondria of cultured cells on a 4000 QTRAP hybrid triple quadrupole/ion trap mass spectrometer equipped with a robotic nano-electrospray source (Advion Triversa NanoMate) for infusion. CL can be quantified as the 13C (m+1) isotopomers of deprotonated doubly charged molecular ions. The isotopomer peaks [M-2H+1]2- or [M-2H+3]2- of doubly charged CL species are very unique, whereas [M-2H]-2 and [M-2H+2]2- may be overlapping with molecular ions in other lipid classes. Therefore, specific CL quantitation is performed by comparison of the de-isotoped peak intensity of the [M-2H+1]2- peak with the de-isotoped peak intensity of the [M-2H+1]2- peak of internal standards (1). In this work, we have used the enhanced resolution (ER) (250 amu/s) ion trap scanning mode of a 4000 QTRAP MS to produce baseline resolved [M-2H+1]2- peaks of CL molecular species, enabling us to execute quantitations down to the fmol/µl level. A software application was written to extract CL 13C (m+1) isotopomer data from MS peak lists. The method was checked by diluting bovine heart CL (Sigma) in a total cellular lipid extract, thereby reproducing the naturally occurring background of a mitochondrial extract (CL are representing 5-20 mol% of the mitochondrial lipids). We have shown the linearity of our assay down to below 1 mol % of CL versus total lipids. For characterization of CL species, we have used the MRM mode of the 4000 QTRAP MS for selective induction of product ion scanning of their 13C (m+1) isotopomers. Using these methods we were able to monitor the effect of Soraphen A, a lipogenic inhibitor, on the mitochondrial CL composition of LNCaP cells. This demonstrates that the method can be applied to quantify CL in biological mitochondrial extracts and to fingerprint their expression profiles after different cellular treatments. 1.Han, X., et al. Journal of Lipid Research (2006), 47, 864-79.