MALDI-TOF-MS Analysis of Lysolipids

LPA was extracted from the unilateral dorsal half of the lumber (L4-L6) spinal cord (6.15 mg tissue weight), as reported previously [13 (link), 19 (link), 20 (link)]. The final sample was dissolved in 50 μl of methanol containing 0.1% aqueous ammonia for MALDI-TOF-MS analysis. One μl sample was spotted on a MALDI plate (Bruker Daltonics, Inc., CA, USA). Immediately, 1 μl of 2’,4’,6’-trihydroxyacetophenone monohydrate solution (10 mg/ml in acetonitrile) was layered on the mixture as matrix solution. After drying, the sample was applied to Ultraflex-I^™ TOF/TOF systems (Bruker Daltonics, Inc., CA, USA). Mass spectrometry was performed in the positive mode, using an accelerating voltage of 25 kV. The laser was used at energy of 30-50% (3.0-5.0 μJ) and a repetition rate of 10-Hz. The mass spectra were calibrated externally using Peptide calibration standard (Bruker Daltonics, Inc., CA, USA). Each spectrum was produced by accumulating data from 150 or 300 consecutive laser shots. Standard of 18:1-LPA was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), while standards of 16:0-, 17:0- and 18:0-LPA were obtained from Doosan Serdary Research Laboratories (London, ON, Canada).