ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE. SelexION™ Technology for Lipid Analysis: Pushing the Boundaries of Lipidomics Baljit Ubhi, Ph.D ASMS Baltimore, June 2014

Merged lipidomic dataMultiple data analysis tools Unraveling the Multiple lipidomic platforms identification of Biomarkers Liquid-liquid extraction Data Processing Interpretation & Reporting Lipidomic Analysis flLJR LipidView ■ btimlW LJfad Oianclart Stahlman, M. et al J Chromatogr B Analyt Technol Biomed Life Sci. 2009

Comprised of Multiple, Distinct Structural Lipid Classes Fatty Acids Steroids Lipid Terpenes Vitamins Mono-, Di- and Waxes Sphingolipids Triglycerides Glycerophospholipids I Sphingomyelins Ceramides Cerebrosides Gangliosides Ether Phospholipids Activating Plasmalogens Diacyl-Linked Phospholipids Oxidized Phospholipids Halogenated Lipids Lipids play an essential role in human physiology: • Metabolic homeostasis • Cell signaling • Cell and organelle structure And disease: • Inflammation • Cancer • Cardiovascular disease • Diabetes • Inflammatory bowel disease • Neurological diseases

• Lipidomic spectra are incredibly complex • MS/MS spectra generated on precursors in zones of isobaric overlap will contain product ions from other isobaric species

SelexION™ Technology Ion Mobility Device Components 1. Orifice Plate • Robust, easy-to-install, hardware components: ‒ ‒ ‒ ‒ 5 No tools required No cables No need to break vacuum Installation in about 2 minutes

How Does DMS Separate Ions? Differential Mobility Spectrometry (DMS) is the term used for planar geometry Gas/Modifier flow Separation waveform (SV): radially displaces ions towards one or the other electrode, depending upon high and low mobility characteristics 6 Compensation voltage (COV): restores the trajectory for a given ion or range of ions to allow them to transmit through the DMS device and enter the mass spectrometer

SelexIONTM Technology Separates Phospholipid Classes Experiment: MRM Scan of 6 Phospholipid Standards with COV Ramp • Using DMS alone, a mixture of lipids can be separated into it’s individual components • Baseline separation can be achieved, completely abrogating isobaric interference Proof of concept: DMS separates simple lipid mixtures Implications: “Cleaner” quantitative data using mrm scan modes (esp., MRM HR); Improved qualitative analysis 7

Effects of DMS on Complex Lipid Analysis Can DMS help resolve lipids from a complex, biological lipid mixture? Experiment 1: EMS Scan of Bovine Heart Extract; No DMS 723.70 7e6 6e6 5e6 Experiment 2: EMS Scan of Bovine Heart Extract; DMS with COV Ramp 8.3 -1.5

An individual phospholipid subclass can be extracted from an EMS scan based on its differential COV • Simplifies LipidView™ Software searching • Increases confidence in lipid identification

Relationship Between Dipole Moment and COV 8 4.5 5 Model headgroup dipole moment (D) Isopropanol as a chemical modifier in DMS 10

Lipid Analysis by AB SCIEX TripleTOF® 5600+ System • Mass Range ‒ Q1 selection – 5 - 1250 m/z ‒ TOF MS – 5 – 40 000 m/z • Speed ‒ 10 ms minimum accumulation times ‒ Up to 100 MS/MS scans per second in IDA • Resolution ‒ High resolution mode ~30,000 ‒ High sensitivity mode ~15,000 • Mass Accuracy ‒ 1-2 ppm RMS (external calibration)

TOF MS Analysis of Lung Lipid Extract Using DMS Phospholipids; Cardiolipin Fatty Acids Experiment: TOF MS with COV ramp in negative ion mode; Infusion of 100 μg/mL lung lipid extract 12

Experiment: TOF MS with COV ramp in negative ion mode; Infusion of 100 pg/mL lung lipid extract > XIC from DMS Expenmenls.wit«sample 12) - S«mple5X-2 DMSon COVromp DR=44 NtG-2.5a

DMS Resolves Fatty Acid Cis/Trans Isomers Experiment: TOF MS with COV ramp in negative ion mode; Infusion of 100 μg/mL lung lipid extract Resolution of cis/trans isomers usually requires chromatographic separation prior to analysis; DMS can remove this need, increasing the speed of FA analysis

11C trom UMb bxpenments.wm (sample 14) - bample pa-4 UMb on CUV ramp UK=44 NbG 4.0 acc, -1 Uh Mb i.lUU - 4UUU) Spectrum from DMS Experiments.wiff (sample 12) - Sample 5X-2 QMS on COV ramp DR=44 NEG “ 2.5 acc, -TOF MS (100 - 2000) from 13.658 to 13.827 min

f£J A ♦ ’ % A 1a w ’ «-tA I QSSlStf Spectrum from DMS Experiments.wiff (sample 6) - Sample 5X-2 DMS on COV ramp DR=42.5, +TOF MS (100 - 2000) from 3.517 to 3.740 min COV = 0.4 to 1.7 V

Resolution of Triglycerides from Complex Mixtures of Lipids COV for TAG

SLICE: Structural Lipidomics Investigations using Chemical Effects • Molecules are separated in CV space prior to entering the MS • Chemical modifiers enhance compound resolution DMS-Separated Precursors • Minimizes isobaric overlap MS/MS of Selected Precursors

Confident Identification of Isobaric Compounds Experiment: DMS MS/MSALL (NEG) of bovine heart extract © 2014 AB SCIEX Duchoslav E., Campbell, J. L., Baker, P.R.S., Patterson B.T., ASMS 2013, ThP 28-586

Comparison of lipid profiles obtained with and without DMS separation * Species with 3 or more chains (TAG, CL) are represented as sum of all permutations

SelexION™ Technology for Lipidomic Analysis Effective lipid resolution without a need for complicated chromatographic strategies • SelexION™ Technology isolates lipid classes prior to MS/MS analysis, which reduces isobaric and isotope overlap and enables accurate identification and quantitation • Lower LOQ/LOD for targeted lipidomics; reduction in matrix interference

Zora Biosciences •Kim Ekroos •Tuulia Sylvanne Wollongong University • Todd Mitchell • Steven Blanksby • Simon Brown • Al Maccarone UCSD • Ed Dennis • Oswald Quehenberger • Paul Norris • Aaron Armando Eli Lilly • Phil Sanders AB SCIEX • Paul Baker • Larry Campbell • Michael Jarvis • Eva Duchoslav • Brigitte Simons • Christie Hunter • Baljit Ubhi Avanti Polar Lipids LipidMAPS.org