A relative quantitative positive/negative ion swit

Authors: Susanne B Breitkopf Stéphane J H Ricoult Min Yuan Ying Xu David A Peake Brendan D Manning John M Asara

Publish Date: 2017/02/07

Volume: 13, Issue: 3, Pages: 30-

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Abstract

Here we present an untargeted 30 min LCMS/MS platform that utilizes positive/negative polarity switching to perform unbiased data dependent acquisitions DDA via higher energy collisional dissociation HCD fragmentation to profile more than 1000–1500 lipid ions mainly from methyltertbutyl ether MTBE or chloroformmethanol extractionsThe platform uses C18 reversedphase chromatography coupled to a hybrid QExactive Plus/HF Orbitrap mass spectrometer and the entire procedure takes ~10 h from lipid extraction to identification/quantification for a data set containing 12 samples ~4 h for a single sample Lipids are identified by both accurate precursor ion mass and fragmentation features and quantified using LipidSearch and Elements softwareUsing this approach we are able to profile intact lipid ions from up to 18 different main lipid classes and 66 subclasses We show several studies from different biological sources including cultured cancer cells resected tissues from mice such as lung and breast tumors and biological fluids such as plasma and urineUsing mouse embryonic fibroblasts we showed that TSC2−/− KD significantly abrogates lipid biosynthesis and that rapamycin can rescue triglyceride TG lipids and we show that SREBP−/− shuts down lipid biosynthesis significantly via mTORC1 signaling pathways We show that in mouse EGFR driven lung tumors a large number of TGs and phosphatidylmethanol PMe lipids are elevated while some phospholipids PLs show some of the largest decrease in lipid levels from ~ 2000 identified lipid ions In addition we identified more than 1500 unique lipid species from human blood plasmaWe thank the Daniel Tenen lab at BIDMC for providing frozen lung tissue We also thank Simon Dillon and Towia Libermann at BIDMC for providing human plasma samples This study was funded by grants from the National Institutes of Health 5P01CA120964 BDM and JMA 5P30CA006516 JMA and R35CA197459 BDM from the National Science Foundation DGE1144152 SR and the BIDMC Research Capital Fund for funding the mass spectrometry instrumentation JMAJMA YX SBB SR and DP developed the platform JMA MY SR and SBB wrote the protocol DP and BM edited the protocol and provided insight YX SR SBB and MY prepared biological samples for testing the protocol JMA MY SB and SR analyzed data

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