Agilent j w gc column db 1

The PLs prepared as described above were hydrolyzed with Phospholipase A2 (PLA2, P6534, Sigma, St. Louis, MO). The resulting sn-2 fatty acyl groups were extracted by the Dole’s method [34 (link),43 (link),44 (link)] and analyzed by gas chromatography–mass spectrometry [GC–MS, Clarus 680 gas chromatograph interfaced with Clarus SQ 8C mass spectrometer (Perkin Elmer, Wellesley, MA) equipped with an Agilent J&W GC column DB-1 (Agilent Technologies Inc., Santa Clara, CA, USA)], as described previously [45 (link)]. Lysophospholipids (LPLs) were analyzed using the total lipid extracts from an aliquot of the PLA2 reaction product by ESI–MS as described above.

Phospholipids were extracted from EMVs and the cells with methanol/chloroform (2:1, vol/vol) by the Bligh and Dyer procedure (Bligh and Dyer, 1959 (link)). The extracts were analyzed by ESI-MS with a triple-quadrupole Sciex API 3000 LC/MS/MS System (Applied Bio-Systems, Foster City, CA, United States) equipped with an ionspray ion source in the negative mode (Cho et al., 2012 (link)). The ionspray voltage was −4,200 kV. The spectra were recorded in the range of m/z 600–800. For fatty acyl chain analysis, the phospholipid extracts were methyl-esterified as described previously (Tokunaga et al., 2017 (link)). The methyl-esterified samples were analyzed with a Clarus 680 gas chromatograph coupled with a Clarus SQ 8C mass spectrometer (Perkin-Elmer, Wellesley, MA, United States) equipped with an Agilent J&W GC column DB-1 (Agilent Technologies, Inc., Santa Clara, CA, United States).