Principles and Practice of Mass Isotopomeric MultiOrdinate Spectral Analysis (MIMOSA) to Assess Metabolic Flux"

Transcription

1 Principles and Practice of Mass Isotopomeric MultiOrdinate Spectral Analysis (MIMOSA) to Assess Metabolic Flux" Richard G. Kibbey M.D., Ph.D. Associate Professor Departments of Internal Medicine and Cellular & Molecular Physiology Yale University School of Medicine

2 What is wrong with my metabolism??!? TCA rate RPM x

3 The KREBS Cycle

4 Canonical Insulin Secretion Glucose GLUT Glucose Insulin Release + Ca GK [Ca + ] i VDCC cytoplasm NADPH Pyruvate SCS-GTP NADH Congenital Hypoglycemia TCA mitochondria OXPHOS O CO HHF6 (GLUD1) ATP ADP K + K ATP

5 MIMOSA was developed to reconcile inconsistancies in stable isotope analysis How to measure glucose oxidanon in insulin secrenng cells? 1 [U- 13 C 6 ]Glucose Acetyl-CoA Citrate Glutamate 13 C - Enrichment (%) ? PEP M+3 Glutamate M+

6 Glycolysis Pyruvate Kinase Pyruvate Dehydro -genase Pyruvate Carboxy -lase Citrate Synthase Isocitrate dehydro -genase P P P P Aldolase Fumarase PEPCK P P Succinyl CoA Synthetase OGDH N N Trans Aminase P P P Gluco Kinase Mitochondria

7 Glycolysis Pyruvate Kinase Pyruvate Dehydro -genase Pyruvate Carboxy -lase Citrate Synthase Isocitrate dehydro -genase P P P P Aldolase Fumarase PEPCK P P Succinyl CoA Synthetase OGDH Trans Aminase N N P P P Gluco Kinase Mitochondria

8 Concentration-Based Flux Measurements Concentration Flux Metabolite X

9 Isotope Enrichment-Based Flux Measurements 4% Enriched Enrichment Flux 5% Enriched

10 Steady state enrichments flux Product Enrichment depends on precursor enrichment V A V B V C (%) 5 13 C (%) 5 13 C (%) 5 f A (V) f A (4V) f A (V/) Time f B (V) Time f B (4V) Time f B (V/)

11 The Tyranny of reversible reactions

12 Where to live? Fate map Flux map

13 TCA Jam: Cycles are not Rings

14 MIMOSA Mass Isotopomer Multi Ordinate Spectral Analysis MIMOSA Platform Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle Graphical Abstract Authors Resource Tiago C. Alves, Rebecca L. Pongratz, Xiaojian Zhao,..., Gary W. Cline, Graeme Mason, Richard G. Kibbey Correspondence In Brief Quantitative assessment of intracellular metabolism requires measuring the enzyme-to-enzyme flow of metabolites. Mitochondria have multiple nodes where metabolites intersect, scramble, and diverge, complicating isotope labeling. Alves et al. use LC-MS/MS to decipher step-wise position-specific transfer of 13 C coming from glucose into subsequent metabolites through glycolysis and around the TCA cycle.

15 A Logues vs. Mers Isotopologues: Position Non-Specific e.g., Citrate M+3 (/64 potential combinations) Isotopomer: Position Specific 8 species Oxidative Anaplerotic 3 families

16 Metabolism of glucose OAA Glutamate Citrate j OAA Glucose OAA Citrate Glutamate OAA i Glutamate Glutamate g Citrate h Citrate f d TCA IDH f CS TCA IDH c OAA Pyruvate Citrate Glutamate OAA a PC PDH IDH 1 TCA 1 CS b IDH TCA CS e IDH TCA b k

17 Position from MS/MS data Originated from Acetyl-CoA Detected by MS Originated from OAA M+ M+1 M+ M+3 M+4 M+5 146/41 147/41 147/4 148/41 148/4 148/43 149/41 149/4 149/43 15/4 15/43 151/43

18 MIMOSA: Leveraging Position pyruvate β-oxidation aspartate AcCoA OAA citrate malate Φ α-ketoglutarate glutamate succinate

19 Mapping Substrate Flows (PEP) Φ POCit Φ CitG1* Citrate Cit a,d, f,h,i, j glutamate Glut C 4,5 +! h Φ CitG Citrate Cit# glutamate + i " 4 + j $ & % Aspartate((D)( Pyruvate( Φ! Φ CitG3 Citrate PAc% Cit g + h + c (i + e) $ # + + j& glutamate " 4 % Φ OP% Acetyl7CoA(! c Φ AcCit% Φ CitG4 Citrate Cit# glutamate + e " 4 + g $ & % Malate( Citrate( Citrate [U 13 C 5 ]Glutamate Glut C1,,3 +3 [1,,3 13 C 3 ]Glutamate! b + f Φ CitG5 Citrate Φ OAA( Cit glutamate MOD% Φ + h + c 3(i + e) $ # + & Glut " 4 4 % C1,,3 + OCit % Φ Φ CitG6 Citrate MO%! b + f Cit glutamate + 3d $ # (19 / 68) & Glut " % C1,,3 +1 aspartate'(d)' malate' OAA' pyruvate' Φ CitM' AcCoA' Φ CitS' succinate' β9oxida<on' citrate' Φ CitG' α ketoglutarate''' ' glutamate' Φ CitG7 Citrate Cit( (b, c, d, e, f, g, h,i, j, 3 (19 Φ CitG% / 68))) glutamate Glut C1,, Glut C1,,3 + + Glut C1,,3 +3 Φ SM% Φ GS% Φ * **.76 Φ PAc Φ AcCit Φ CitGΦGS Succinate( CitG (1) CitG () CitG (3) CitG (4) CitG (5) CitG (6) CitG (7) Φ SMΦMO Φ *** Φ OCit' Φ OP Glutamate( CitS(1) CitS() CitS(3) CitS(4) Φ CitSM(1) CitSM() CitSM(3) Alves et al. Cell Metabolism 15

20 Isotopomers vs. Isotopologues Calculated vs. Actual Pyruvate Carboxylase Pyruvate Φ(1) = " + 3 "%&%'( (*+) " + 3 *-./%'( " + 3 "%&%'( (*+) V PEPCK M+3 (Succinate) V ME V PC M+3 (PC) Malate Acetyl-CoA V CS Succinate V PC /V CS (Calculated Output) 1 1 V PC /V CS (Model Input) Φ() Φ(3) = Φ(4) Φ(5) = " + 3 "%&%'( " + 3 )*+,-%'( " + 3 "%&%'( " + 3 *+,,-.%'( " + 3 /1+%'( = " + 3 "%&%'((*+) " + 3 *-./%'( = " + 3 "%&%'( " + *+',%'( Φ(6) = " + 3 %&'()'* + " + 5 %&'()'* " + 3,-(./)'*

21 Change of Perspective PDH Pyruvate Oxidation PC Pyruvate Carboxylation Alves et al. Cell Metabolism 15

22 1 Fit to mathematical model: 5 CWave Cit a [1,3,6,4,5-13 C 5 ]Citrate. 1.5 F 1. Pyruvate M + G Sensitivity to.5 mm change in glucose Glutamate Total C concentration Succinate M+4 Malate M ! Cit f [U- 13 C 6 ]Citrate Succinate M [1,,3,4,5-13 C 5 ]Citrate Cit a, f, i, d, h, j Malate M+3 1 [,3,4-13 C 3 ]Succinate [,3,6,4,5-13 C 5 ]Citrate [4,5-13 C ]Glutamate [(1,,3)(1,3,4)(1,,4)- 13 C 3 ]Succinate [U- 13 C 4 ]Succinate Malate M+ [1,,3-13 C 3 ]Malate [1,,4-13 C 3 ]Malate Isotopologue Fit SD Positionless analysis Isotopomer Fit severely misses [U- 13 C 4 ]Malate OAA M+ [1,,3-13 C 3 ]OAA [1,,4-13 C 3 ]OAA anaplerosis! PC Flux (µm/µm Taurine/min) 4 1 Time 5 (min) Isotopomer Fit Isotopologue Fit isotopologue and isotopomer data [U- 13 C 4 ]OAA 1 8 data for citrate, glutamate, 6 [1,,3-13 C 3 ]Citrate [1,- 13 C ]Citrate Isotopomer Fit succinate and Isotopologue Fit cles correspond to the enrichment data Frequency SD !"# calculated using the isotopomer and cetyl-coa: (C) [1,,3-13 C 3 ]citrate and (D) µm / µm Taurine / min.1. Time C D E F PDH CS PC Alves et al. Cell Metabolism

23 MIMOSA Mass Isotopomer Mul?-Ordinate Spectral Analysis PEP [U- 13 C 6 ]Glucose V PK V PEPCK V ME Pyruvate Fa]y Acids V PDH V PC Acetyl-CoA V CS V β-oxidanon Malonyl-CoA V Lipogenesis Citrate OAA V ICDH Malate αkg V X Glutamate Alves et al, Cell Metab 15 V SDH Succinate Glutamine V Glutaminase

24 Pyruvate Carboxylase (PC) is the most responsive flux to changes in glucose concentranons in INS-1 Insulin (ng/hr/mg protein) P <.5 P <.1 G.5 G5 G7 G9 Fluxes (Normalized to G.5) V PC R =.957 V PDH R = Insulin (Normalized to G.5) Fluxes (µm / µm Taurine / min)..1. G.5 G5 G7 G9 Alves et al, Cell Metab 15

25 MIMOSA Workflow Integration (MS/MS) Φ (Steady state) ν (dynamic) P/D Natural Abundance Isotopomer Deconvolvement

26 Caveat Emptor Concentration Flux Enrichment Flux Fate map Flux map Cycles Rings Steady State Kinetic Exponential fit single point TCA turns more than once Reversible reactions matter Location, Location, Location MIMOSA: not just for brunch

27 MIMOSA: high sensinvity of mass spectrometry with the posinon specificity of 13 C-labeling Glucose PEP Pyruvate Raw Mass Spec M+ 1 st turn TCA nd turn TCA vs Acetyl-CoA M+3 PDH Labeled Acetyl-CoA Unlabeled Acetyl-CoA OAA V CS Citrate M+4 vs Malate Succinate αkg M+5 PC Label M+6 vs TCA Label

28 THANK YOU!! Abudukadier Abulizi Tiago Alves Rebecca Cardone Gary Cline Joelle Hillion Selin Isguven Sean Jesinkey Anila Madiraju Graeme Mason Rachel Perry Raaisa Doug Rothman Stephan Siebel Gerald Shulman Romana Stark Bei Wang OrLando Yarbarough Xiaojian Zhao Lingjun Ma TCA rate RPM x

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