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Systems Biology -

U sing Mixed Isotope Labeling for Systems Biology Shane Lamos Vermont Genetics Network Annual Retreat August 7, 2013. Systems Biology -. Seeks to discover the link between molecules and physiology Top Down – “ omics ”, phenomenon based Bottom Up – mechanism based

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Systems Biology -

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  1. Using Mixed Isotope Labeling for Systems BiologyShane LamosVermont Genetics Network Annual RetreatAugust 7, 2013

  2. Systems Biology - Seeks to discover the link between molecules and physiology Top Down – “omics”, phenomenon based Bottom Up – mechanism based e.g. enzyme kinetics

  3. Systems Biology – Top Down

  4. Research Projects • Develop new chemical methodologies to explore • biological systems • Mixed Isotope labeling coupled with MS • Proteome • Identification of small molecule protein targets • Enhanced detection of proteins with charge auxiliaries • Metabolome • Relative quantification of amine metabolites • Relative quantification of fatty acid metabolites • Multiplexed analysis of carboxylic acid metabolites • Relative quantification of small carboxylic acids

  5. L L L H H H Isotope Dilution vs Isotope Labeling Experimental Metabolites Internal Standard Experimental Metabolites Control Metabolites Label with Light (L) Reagent Label with Heavy (H) Reagent ?

  6. How Does Relative Quantification of Metabolites Work? Sample A Sample B Mix Samples x x y y x x y y Light Isotope Label Heavy Isotope Label Separate Analytes Electrospray MS x x y y Y Y X Intensity X m/z

  7. Cholamineas an Isotopic Label for Fatty Acids • Fixed positive charge enhanced detection in positive ion-mode (MS) • Isotopically labeled metabolites co-elute • Light and heavy label signifies carboxylic acid functionality Light M + 0 Heavy M + 9

  8. Isobaric Cholamines

  9. Enhanced Detection of Labeled Metabolites • Ionizability of Analyte is Enhanced Valine, Asparagine, Glutamic Acid, Tryptophan

  10. Extracted Ion Chromatograms of Co-Eluting Heavy- and Light-Labeled Carboxylic Acids 16:0 18:1 (Fatty Acid 18:2) 18:2 18:0 16:1 20:4

  11. Model Egg Labeling with Cholamine Sample A Sample B Limits of Detection (LOD) = 10 – 30 fmol

  12. Relative Quantification of Chicken Egg Fatty Acids Sample A (CLA + Olive Oil) Sample B (Control) Mix Samples x x y y x x y y Light Isotope Label Heavy Isotope Label Separate Analytes Electrospray MS x x Y y y Y X Intensity X m/z

  13. Quantification of Fatty Acids From Chicken Eggs (CLA + OO vs Control) (Juvenile Egg) 16:0 + 18:0 16:1 + 18:1 (Fertile Egg) CLA (18:2) SCD-1

  14. Quantification of Fatty Acids From Other Chicken Egg Diets

  15. x x x y y y Multiplexing Sample B Sample A Sample C Medium Isotope Label Light Isotope Label Heavy Isotope Label x x x y y y X X x x y Intensity X x y y m/z

  16. Multiplexing Isobaric Cholamines

  17. Simulated Multiplexed Labeling Sample A Sample B Sample C

  18. Multiplex LabelingCaged vs Cage Free

  19. Five-Plex Isobaric Cholamines

  20. Five-Plex Isobaric Cholamines Using a silkworm hyperglycemic model of type II diabetes Silkworms grown under 5 different diet scenarios Measuring free fatty acids in hemolymph simultaneously

  21. Cholamines as Isotopic Labels Effective for relative quantification of carboxylic acids Pre-ionized fatty acids readily quantified in LC-MS Heavy- and light-labeled analytes co-elute “Multiplexing’ of carboxylic acid functionality

  22. Hydrophobic Tag for Small Carboxylic Acids Many important small carboxylic acid metabolites Too polar for chromatographic retention labeled with cholamine Need a more hydrophobic mixed isotope labeling reagent

  23. Synthesis of DMP and DMP + 6 Non-Isotopic and Isotopic Putrescine (13C0 and 13C4) Non-Isotopic and Isotopic Formaldehyde (H212CO and H213CO) DMP {M + 0, and M + 6} Labeled a series of small carboxylic acids with DMP {0, and 6} Investigating coupling conditions for precision and accuracy Also using DMP for enhanced ETD Top-down Proteomics

  24. Collaborators: Dr. Ying Wai Lam Director UVM/VGN Proteomics Facility Dr. Lloyd M. Smith UW-Madison Group Members: Jon Downey ’09 Andrew Therrien ‘12 Zach Eldridge ’10 Christopher Dustin ‘12 Katie Summo’10 Heidi Chapman ‘13 Jeff Dukette ’11 Emily Dieter ‘14 Derrick Cumberbatch ’11 Wes Cubberley ‘14 Gus Torde ’11 *Katie Schutt ‘14 *Sam Drajesk ‘15 Acknowledgements Support: Vermont Genetics Network Saint Michaels College Gianni Fund and VPAA Fund

  25. Saint Michael’s College

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