Metabolomic and Lipidomic Research in Diabetes and Obesity 08/07/2013
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1 Novel Tools for omics -based Analysis of Complex Mixtures; Application to a Investigation of the Effect of Prolonged Glucose Stimulation on the Lipid Profile of Mouse Heart Muscle John P. Shockcor, PhD, FRSC CChem Director of Strategic Operations, Pharmaceutical and Life Sciences, Waters Corp. Professor, Dept. Of Surgery and Cancer, Imperial College, London, UK Visiting Fellow, Dept. of Biochemistry, University of Cambridge, UK 2012 Waters Corporation 1 Metabolomic and Lipidomic Research in Diabetes and Obesity Prof. Jules Griffin, MRC HNR & University of Cambridge Dr.Helen Atherton Dr.Melanie Gulston 1
2 Effect of insulin on glucose uptake and metabolism. Insulin binds to its receptor (1) which in turn starts many protein activation cascades (2). These include: translocation of Glut-4 transporter to the plasma membrane and influx of glucose (3), glycogen synthesis (4), glycolysis (5) and fatty acid synthesis (6) Waters Corporation 3 The Diabetic Heart Type II diabetes is characterized by insulin resistance, and in the heart this results in a failure to recruit Glucose Transporter 4 (GluT4) into the cell membrane during periods of high blood glucose. As a result, metabolism of the diabetic heart has an increased reliance on beta-oxidation of fatty acids. This has been linked to the increased lipid load which may result in lipotoxicity in the diabetic heart. In addition the ATP generated by fatty acid oxidation requires more oxygen compared with that derived from the oxidation of glucose, increasing the risk of ischaemic heart disease Waters Corporation 4 2
3 Study Description In this study we have examined a mouse model where Glucose Transporter 1 has been increased in expression selectively in the heart. GLUT1 is responsible for the low-level of basal glucose transport. It is a non-insulin dependent glucose transporter and as such it will dramatically increase glucose metabolism in the heart Waters Corporation 5 Study Description Wild-type and transgenic mice were sacrificed at 3 months and the heart tissue rapidly dissected and stored at -80C. A chloroform/methanol extraction was performed and the lipids in the organic layer for all samples were profiled using a UPLC/Synapt G Waters Corporation 6 3
4 Question? What is the effect of prolonged glucose stimulation on the lipid profile of heart tissue? 2012 Waters Corporation 7 Intact Lipid Analysis: 2012 Waters Corporation 8 4
5 Chromatography 2012 Waters Corporation 9 Direct Infusion of a Tissue Extract 2012 Waters Corporation 10 5
6 Exapnded Region around m/z 806 PC 38: Waters Corporation 11 TIC of Tissue Extract 2012 Waters Corporation 12 6
7 TIC and XIC for m/z PC 38:6 PC 16:0/22:6 Isotope of 804 PC 18:2/20: Waters Corporation 13 UPLC and MS Conditions for Global Lipid Profiling 2012 Waters Corporation 14 7
8 UPLC/MS from liver extract PC, PG, PE ChoE & TG lysophospolipids SM, DG FFA PG,PI, PS, PE 2012 Waters Corporation 15 Trizaic 2012 Waters Corporation 16 8
9 TRIZAIC UPLC System 2012 Waters Corporation 17 nanotile Flow Path 2012 Waters Corporation 18 9
10 Free Fatty Acids 18:0 18:1 16:0 18:2 20:4 22:6 18:3 Linear response between ng/ l and 5 ng/ l Mobile phase A: ACN/Water (60:40) with 10 mm NH 4 formate and 0.1% FA Mobile phase B: IPA/ACN (90:10) with 10 mm NH 4 formate and 0.1% FA 2012 Waters Corporation 19 Liver Extract: Positive Ion Mode Column: CSH C18, 0.15 x 100 mm TG PC, SM, PE, PG, PI LP 2012 Waters Corporation 20 10
11 Total Lipids Extract from Liver Negative Ionization mode PC, SM, PE, PG, PI FFA, LP 2012 Waters Corporation 21 Tic of Human Urine (Positive Ion) 2012 Waters Corporation 22 11
12 UPC Waters Corporation 23 UPC 2 Free Fatty Acid FFA Mix 1 FFA Method 0.5ul inj 15CV0 17:14:13 TAGs_ _ % C14: C16: C18: C20: C22: C24: Method Conditions: 03-Aug : TOF MS ES- BPI Instrument: 1.71e5 ACQUITY UPC 2 Column: ACQUITY UPC 2 HSS C 18 SB Co-solvent: MeOH w/ 2g/L Ammonium Formate Gradient: 1 to 10% over 5 minutes* Flow: 2.5 ml/min Temperature: 60 0 C CCM Pressure: 1885 Injection Vol: 0.5uL Make-up flow: 0.2mL/min of 0.1% formic acid Splitter: Upchurch cross 1/16 PEEK C12: Sample Concentration: 0.25mg/mL FFA: C8 to C24 0 C8:0 C10: Time Waters Corporation 24 12
13 UPC 2 /oa-qtof: 2012 Waters Corporation 25 MS Analysis 2012 Waters Corporation 26 13
14 Electro-Spray Ionization 2012 Waters Corporation 27 A Quadrupole Time-of Flight Mass Spectrometer 2012 Waters Corporation 28 14
15 Fragmentation of a Typical Glycerophospholipid O sn-1-h20 O sn-1 P R 1 O O O HO R 2 O H sn-2-h20 N + O sn-2 Phosphotadylcholine (PC) 2012 Waters Corporation 29 Fragmentation of Triglycerides sn-2-nl NL sn-1 O sn-1-nl NL sn-3 O R 1 O R 2 O R 3 O H O sn-2 sn-3-nl Note: The fragmentation for Mono and Di Glycerides cleaves the same bonds Waters Corporation 30 15
16 UPLC/MS E (Step 1) 2012 Waters Corporation 31 UPLC/MS E ( low energy) Co-Eluting Ions Ionized Precursors Precursors Transferred to TOF MS 2012 Waters Corporation 32 16
17 UPLC/MS E (high energy) Co-Eluting Ions Ionized Precursors Precursors & Products Transferred to TOF-MS 2012 Waters Corporation 33 UPLC/HDMS E (low energy) deconvoluting chimericy Co-Eluting Ions Ionized Precursors Precursors IMS Separated Precursors Transferred to TOF MS 2012 Waters Corporation 34 17
18 UPLC/HDMS E (high energy) deconvoluting chimericy Co-Eluting Ions Ionized Precursors Precursors IMS Separated Precursors & Products Time Aligned 2012 Waters Corporation 35 UPLC/HDMS E 2012 Waters Corporation 36 18
19 Fragmentation Spectrum of TG (18:0/18:1/18:1) m/z Waters Corporation 37 Export to EZinfo 2012 Waters Corporation 38 19
20 PLS-DA of All Groups 2012 Waters Corporation 39 Transfer of Loadings 2012 Waters Corporation 40 20
21 Tagging Imported Loadings 2012 Waters Corporation 41 Creating a Filter 2012 Waters Corporation 42 21
22 Working with Selected Compounds Mined from MVA 2012 Waters Corporation 43 Acknowledgements Waters Corporation Milford, USA Scot Germanos Giorgis Issac Andy Baker Waters Corporation Manchester, UK James Langridge Mike Morris John Hoyes Martin Green Jason Wildgoose Kevin Giles Steve Pringle Ronan O Malley Alistair Wallace Jeff Goshawk Richard Gilpin 2012 Waters Corporation 44 22
23 Acknowledgements University Of Cambridge Cambridge, UK Jules Griffin Helen Atherton Melanie Gulston Imperial College London, UK Jeremy Nicholson Elaine Holmes Liz Want Matt Lewis Merck USA Jose Castro Perez 2012 Waters Corporation 45 Acknowledgements 2012 Waters Corporation 46 23
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