Seppac c18

Peptides were synthesized by the Merrifield solid phase method manually with 9-fluorenylmethoxycarbonyl (fmoc) chemistry on 4-[2,4-dimethoxyphenyl-N-(9-fluorenylmethoxycarbonyl)aminomethyl]phenoxy resin [27 (link)]. After cleavage from resins, crude peptides were purified using an octadecyl silica (ODS) gel (SEPPAC-C18, Waters, MA). Crude peptides were bound to the ODS gel and washed with 0.1% trifluoroacetic acid (TFA). The peptides were eluted with 60% (v/v) acetonitrile in 0.1% TFA. The eluate was lyophilized and the purified peptide was weighed. The molecular weights of the synthetic peptides were determined using MALDI-TOF mass spectrometry (Autoflex, Bruker Daltonics, Germany). MALDI-TOF mass spectrometry was manually acquired in a positive reflect mode controlled by FlexControl software (Bruker Daltonics, Germany).

Lipids were extracted and isolated as described previously (Zhang et al. 2018a (link)). Briefly, samples were purified through an SPE column (500 mg × 6 mL Sep-Pac C18; Waters, Milford, MA, USA). The targeting components were next dried under N₂ and residues were reconstituted utilizing a methanol and water mixture (v/v, 1:1). A final 10 μL aliquot of each sample was introduced into an AQUITY UPLC (Waters) (BEH-C18 2.1 × 100 mm, 2.1 × 50 mm, 1.7 μm, Waters)-AB SCIEX QTRAP 6500 system (ESI mode). Moreover, the column, gradient program, and multiple reaction monitoring (MRM) were optimized during the monitoring processes (Zhang et al. 2018a (link)). Analyst® 1.6.2 and MultiQuant™ software (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA, USA) were used to acquire and quantify all lipids.