U sing Mixed Isotope Labeling for Systems - PowerPoint Presentation

1. Using Mixed Isotope Labeling for Systems BiologyShane LamosVermont Genetics Network Annual RetreatAugust 7, 20131

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

3. Systems Biology – Top Down3

4. Research Projects4 Develop new chemical methodologies to explore biological systemsMixed Isotope labeling coupled with MSProteomeIdentification of small molecule protein targetsEnhanced detection of proteins with charge auxiliariesMetabolomeRelative quantification of amine metabolitesRelative quantification of fatty acid metabolitesMultiplexed analysis of carboxylic acid metabolites Relative quantification of small carboxylic acids

5. 5HHIsotope Dilution vs Isotope LabelingExperimental MetabolitesInternal StandardLabel with Heavy (H) ReagentLabel with Light (L) Reagent?Experimental MetabolitesControl MetabolitesLLLH

6. 6How Does Relative Quantification of Metabolites Work?Sample ASample BLight Isotope LabelHeavy Isotope LabelMix SamplesSeparate AnalytesElectrospray MSm/zIntensityXYYXyxyxyxyxyxyx

7. 7Cholamine as an Isotopic Label for Fatty AcidsFixed positive charge enhanced detection in positive ion-mode (MS)Isotopically labeled metabolites co-eluteLight and heavy label signifies carboxylic acid functionality Light M + 0 Heavy M + 9

8. Isobaric Cholamines 8

9. 9Enhanced Detection of Labeled MetabolitesValine, Asparagine, Glutamic Acid, TryptophanIonizability of Analyte is Enhanced

10. 10Extracted Ion Chromatograms of Co-Eluting Heavy- and Light-Labeled Carboxylic Acids16:118:216:018:118:020:4(Fatty Acid 18:2)

11. Model Egg Labeling with Cholamine11Sample A Sample BLimits of Detection (LOD) = 10 – 30 fmol

12. 12Relative Quantification of Chicken Egg Fatty Acids Sample A (CLA + Olive Oil)Sample B (Control)Light Isotope LabelHeavy Isotope LabelMix SamplesSeparate AnalytesElectrospray MSm/zIntensityXYYXyxyxyxyxyxyx

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

14. 14Quantification of Fatty Acids From Other Chicken Egg Diets

15. 15Multiplexing Sample ASample C Light Isotope LabelHeavy Isotope LabelyxyxyxyxSample B yxyxMediumIsotope Labelyxyyxxm/zIntensityXXX

16. Multiplexing Isobaric Cholamines16

17. Simulated Multiplexed Labeling 17Sample A Sample B Sample C

18. Multiplex LabelingCaged vs Cage Free18

19. Five-Plex Isobaric Cholamines19

20. Five-Plex Isobaric Cholamines20Using a silkworm hyperglycemic model of type II diabetesSilkworms grown under 5 different diet scenariosMeasuring free fatty acids in hemolymph simultaneously

21. Cholamines as Isotopic Labels21Effective for relative quantification of carboxylic acidsPre-ionized fatty acids readily quantified in LC-MSHeavy- and light-labeled analytes co-elute “Multiplexing’ of carboxylic acid functionality

22. Hydrophobic Tag for Small Carboxylic Acids22Many important small carboxylic acid metabolitesToo polar for chromatographic retention labeled with cholamineNeed a more hydrophobic mixed isotope labeling reagent

23. Synthesis of DMP and DMP + 623Non-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 accuracyAlso using DMP for enhanced ETD Top-down Proteomics

24. Acknowledgements24Collaborators: Dr. Ying Wai Lam Director UVM/VGN Proteomics Facility Dr. Lloyd M. Smith UW-MadisonGroup Members:Jon Downey ’09 Andrew Therrien ‘12Zach Eldridge ’10 Christopher Dustin ‘12Katie Summo ’10 Heidi Chapman ‘13Jeff Dukette ’11 Emily Dieter ‘14Derrick Cumberbatch ’11 Wes Cubberley ‘14Gus Torde ’11 *Katie Schutt ‘14 *Sam Drajesk ‘15Support:Vermont Genetics NetworkSaint Michaels College Gianni Fund and VPAA Fund

25. 25Saint Michael’s College