Metabolomics and Lipidomics Approaches for Biomedical Research and Biomarker Discovery

Transcription

1 Metabolomics and Lipidomics Approaches for Biomedical Research and Biomarker Discovery Giuseppe Astarita Principal Scientist, Health Sciences Waters Corp, USA Waters Users Meeting ASMS 30 May, Waters Corporation 1

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3 Metabolomics Approaches Untargeted Scanning for differences using high-res instruments (semi-quantitative; statistics-based; hypothesis-generating) sn-1 O sn-1-h20 O P R 1 O O O HO R 2 O H sn-2-h20 O sn-2 Phosphotadylcholine (PC) N + Structural Elucidation High-res instruments with fragmentation capabilities (chemical structure) Targeted Monitoring selected ions in high-res or nominal mass (quantitative; sensitive; requires internal standards) In situ; Imaging Scanning along x and y axes using high-res instruments (spatial information; molecular histology)

4 Metabolomics and Lipidomics Separate (Chromatography; Ion Mobility) Measure (Mass Spectrometry) Process and Mine (Informatics and Statistics)

5 Metabolomics in Systems Biology

6 Typical Metabolomics Applications WT/KO cells Transgenic mice Treated mice Human subjects Gene function Diet Environmental exposure Disease PNAS, 2015 PLOS ONE, 2014 J Proteome R, 2014 In Preparation

7 Poster 202 Metabolomics: Untargeted Metabolite Profiling II A mass-spectrometry-based metabolic phenotyping strategy to investigate the molecular response to ionizing radiation 2015 Waters Corporation 10

8 Radiation Exposure Occupational exposure (medicine, manufacturing and construction) Medicine (research, diagnosis and therapy) Airline/Space Travel

9 Nuclear and Radioactive Accidents Hiroshima Nagasaki Others: Chernobyl Three Mile Island, US Goiania, Brazil Kozloduy, Bulgaria Fukushima Daiichi

10 Health Effects of Radiation Exposure Cancer Cardiovascular Disease Cognitive decline

11 Molecular Effects of Radiation Exposure DNA Proteins Lipids/Metabolites

12 Aims of the study 1.Investigate the biochemical mechanisms underlying radiation exposure 2. Determine biomarkers or biosignatures associated with radiation exposure Laiakis E. et al, J Proteome Res Sep 2015 Waters Corporation 15

13 Laiakis E. et al, J Proteome Res Sep Study Design Sham Control Irradiated Blood collection Serum preparation Liquid-liquid Biocrates kit SPE clean up Cryostat sectioning LC/TOF MS LC tandem MS Global Metabolic Profiling Targeted Metabolic Profiling Oxylipin Profiling MS Imaging Endogenous metabolite 1 Internal standard

14 Metabolomics Approaches Untargeted Scanning for differences using high-res instruments (semi-quantitative; statistics-based; hypothesis-generating) sn-1 O sn-1-h20 O P R 1 O O O HO R 2 O H sn-2-h20 O sn-2 Phosphotadylcholine (PC) N + Structural Elucidation High-res instruments with fragmentation capabilities (chemical structure) Targeted Monitoring selected ions in high-res or nominal mass (quantitative; sensitive; requires internal standards) In situ; Imaging Scanning along x and y axes using high-res instruments (spatial information; molecular histology)

15 Metabolomics: UPLC Separation Paglia G.et al. Anal Chem 2014

16 Lipidomics: UPLC Separation PC, SM, PG, PE ChoE & TG lysophospolipids SM, DG, ChoE Free Fatty Acids PC, PG,PI, PS, PE Astarita G., et al. PLOS ONE 2014

17 Damen C., et al. Journal Lipid Research 2014 Orthogonal Coordinates PC, SM, PG, PE lysophospolipids ChoE & TG

18 Ion mobility drift time (µs) Ion Mobility Separation: Hybrid Q-TOF TWIMS Poster 649 Ion Mobility: Small Molecule and Metabolomics The analysis of Bile Acids: Enhancement of specificity using an Ion Mobility-TOFMS based approach Paglia G.et al. Anal Chem 2015

19 Data Processing and Mining Tentative Identifications Database search Individual ion maps 4. Control Radiation Data alignment and peak picking Filtering by ANOVA P value Composite ion map after peak picking ANOVA filtering and Multivariate Statistics Control Radiation Laiakis E. et al, J Proteome Res Sep

20 Database Search identifications using Orthogonal Coordinates HMDB In house database no RT In house database with RT <0.6 min ppm m/z error

21 Database Search using Orthogonal Coordinates HDMS E Co-eluting ions With Ion mobility separation Fragmentation Fragments Transferred to TOF-MS HDMS E Low Energy (FA 20:4) % HDMS E High Energy (FA 20:4) <25 fragments % 200 m/z 1400

22 Global Metabolic Profiling m/z Tentative ID ANOVA (p value) Max Fold Change Method PC(P-18:0/22:6) 5.12E Reversed phase PC(P-16:0/20:5) 1.02E Reversed phase PC(P-16:0/20:4) 2.11E Reversed phase PC(P-18:0/20:4) 2.97E Reversed phase PC(P-18:0/20:5) 3.38E Reversed phase PC(P-16:0/22:6) 6.74E Reversed phase SM(d18:1/16:0) 2.09E Reversed phase Carnitine 1.50E HILIC Phenylalanine 1.53E HILIC

23 Metabolomics Approaches Untargeted Scanning for differences using high-res instruments (semi-quantitative; statistics-based; hypothesis-generating) sn-1 O sn-1-h20 O P R 1 O O O HO R 2 O H sn-2-h20 O sn-2 Phosphotadylcholine (PC) N + Structural Elucidation High-res instruments with fragmentation capabilities (chemical structure) Targeted Monitoring selected ions in high-res or nominal mass (quantitative; sensitive; requires internal standards) In situ; Imaging Scanning along x and y axes using high-res instruments (spatial information; molecular histology)

24 Targeted Metabolic Profiling: Metabolomics kits + Metabolite group No. of metabolites Poster 401 Metabolomics: Quantitative Analysis Improved performance of targeted metabolome analysis with Waters Xevo TQ-S and Xevo TQ-S micro instruments Amino acids and Biogenic amines 40 Acylcarnitines 40 Lyso-phosphatidylcholines 14 Phosphatidylcholines 74 Sphingomyelins 14 Hexose 1 Total 183

25 C o n c e n tra tio n (µ M ) Component 3 (11.5%) C o n c e n tra tio n (µ M ) Laiakis E. et al, J Proteome Res Sep Targeted Metabolic Profiling Control Radiation Component 1 (32.2%) C o n tro l *** Diacyl PCs *** *** *** *** R a d ia tio n *** *** *** Ether PCs *** *** *** C o n tro l R a d ia tio n *** 0 P C a a C 3 4 :1 P C a a C 3 6 :5 P C a a C 3 8 :5 P C a a C 3 8 :6 P C a a C 3 4 :4 P C a a C 3 6 :6 0 P C a e C 3 6 :4 P C a e C 3 6 :5 P C a e C 3 8 :4 P C a e C 3 8 :5 P C a e C 3 8 :6 P C a e C 3 2 :1

26 Data Integration: Molecular Effects of Radiation Aldehydes Plasmalogens Oxidation Diacyl PC PLA 2 Arachidonic acid? Metabolites

27 Eicosanoids: Bioactive Oxygenated PUFAs Omega-6 metabolites: pro-inflammatory Omega-3 metabolites: anti-inflammatory

28 Multiplexed Assay for Eicosanoids Profiling Plasma sample Add internal standard mix Load SPE clean up Inject into UPLC/MS-MS Selected Lipid Internal Standard

29 C o n c e n tra tio n (µ M ) C o n c e n tra tio n (µ M ) Component 3 (24%) Eicosanoids Profiling Control Radiation FA 18:3 FA 20:5 Component 1 (26.9%) C o n tro l R a d ia tio n Omega-6s *** C o n tro l R a d ia tio n Omega-3s * H H T re *** 1 1 -H E T E *** 1 4,1 5 -D ih E T re *** 8 -H E T E *** 1 2 -H E T E ,1 8 -D ih E T E * 1 4,1 5 -D ih E T E *** 9 -H O T re Laiakis E. et al, J Proteome Res Sep

30 Pathway Analysis Omega-6 metabolites: pro-inflammatory Omega-3 metabolites: anti-inflammatory

31 Data Integration: Molecular Effects of Radiation Diacyl PC PLA 2 Arachidonic acid? Metabolites 8-HETE

32 Data Fusion: Biosignature of Radiation Exposure Top metabolites correlated with the irradiated phenotype

33 In vivo responses to total body irradiation (TBI) in patients TBI received prior to hematopoietic stem cell transplant (n=15) Total Body Irradiation Serum Pre, 6hr, 24hr Synapt G2 Si Untargeted Metabolomics Untargeted Lipidomics Progenesis QI Pre ~6hr ~24hr SPE clean up Xevo TQ-S Targeted Metabolomics TargetLynx for quantification Statistical Analysis and pathway assignment Peak Picking Statistical Analysis Slide from Evagelia C. Laiakis Pathway annotation HMDB, KEGG, Lipidmaps Validation through tandem MS or fragment matching through online databases

34 Normalized Abundance Normalized Abundance Preliminary Results in Human Subjects 9,12,13-TriHOME 9,10,13-TriHOME 9-HOTrE 9-OxoODE 9-HODE 9-HpODE 13-OxoODE (13-KODE) 13-HODE 13-HpODE 9,12,13-TriHOME LOX Linoleic acid (LA) CYP450 12(13)-EpOME 9(10)-EpOME 12,13-DiHODE 12,13-DiHOME 9,10-DiHOME Linoleic acid p= Dihomo-gamma-linolenic acid p= LOX PGI Dihomo- -linolenic acid 1 (DGLA) TXB 1 COX 15-HETrE PGH Pre 6hr 24hr TXB 2 12-HHTrE 13,14-dihydro-15-keto PGE 2 PGG 2 TXA 2 PGH 2 bicyclo-pge 2 PGE 2 PGF 2 PGD 2 PGA 2 PGJ 2 15-keto-PGF 2 PGC 2 12-PGJ 2 5-HETE 5-oxo-ETE LTC 4 LTD 4 COX 5-HpETE LTA 4 Arachidonic acid (AA) LOX LXB 4 15-HpETE 15-HETE LXA 4 LTB 4 15-oxo-ETE PGE 1 12(S)-HpETE 12-HETE 11-HETE 8-HETE PGF 1 PGD 1 non enzymatic CYP450 HETEs 5(6)-EpETrE 8(9)-EpETrE 11(12)-EpETrE 14(15)-EpETrE 0 Pre 6hr 24hr PGB 2 Slide from Evagelia C. Laiakis 15-deoxy- 12-PGJ 2 LTE 4 5,6-DiHETrE 8,9-DiHETrE 11,12-DiHETrE 14,15- DiHETrE

35 p(corr)[1]p (Correlation) Conclusions Integration of multi-platform lipidomics data highlighted new biochemical pathways associated with radiation exposure S-Plot (Group 1 = -1, Group 2 = 1) The untargeted lipidomics approach uncovered a differential metabolism for diacyl phospholipids and plasmalogen CoeffCS[2](Group) (X Effects) EZinf o _Liv erpos1 (M4: OPLS-DA) :39:03 (UTC-5) The multiplexed assay identified marked alterations in a subset of pro-inflammatory lipid mediators Fusion of multi-platform data highlighted a metabolomics biosignature associated with exposure to radiation exposure 2015 Waters Corporation 39

36 Methodological References: Untargeted

37 Methodological References: Targeted

38 Acknowledgements Albert Joseph Fornace Evagelia C. Laiakis Thomas Hankemeier Rob Vreeker Katrin Strassburg Jim Langridge Rob Plumb Emmanuelle Claude Jeff Mazzeo Ralf Bogumil Cornelia Roehring Therese Koal 2015 Waters Corporation 43