New Mass Spectrometry Tools to Transform Metabolomics and Lipidomics

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1 New Mass Spectrometry Tools to Transform Metabolomics and Lipidomics July.3.13 Ken Miller Vice President of Marketing, Life Sciences Mass Spectrometry 1 The world leader in serving science

2 Omics & the Central Dogma of Biology DNA Genomics 22, genes What could occur RNA Transcriptomics 1, transcripts What may occur Proteins Proteomics 1,, proteoforms What is occurring Metabolomics 2, compounds Lipidomics 1, species Metabolites Lipids What has occurred 2

3 Addressing the Metabolomics Challenges Developing SOPs for major sample types Innovating LC/MS to improve separation & detection Developing streamlined, workflow-oriented software Building and connecting to new and existing database 3

4 Orbitrap Delivers the Highest Quality Data Better data, Better results! Highest Resolution Best Mass Accuracy Widest Dynamic Range Excellent Sensitivity 4

5 LCMS Metabolomics Offerings Quan / Qual analysis of endogenous molecules of low molecular mass (<1-15Da) Profile Detect, align, and quantify components, use statistical analysis to find key features Thermo Scientific Dionex UltiMate 3 RSLC Thermo Scientific Exactive Series MS Identify Putative ID web or local DB search Confirm ID via MS 2 De novo structural ID via MS n Thermo Scientific Orbitrap Fusion MS Thermo Scientific Q Exactive MS Quantify Rountinely quantify sets of putative biomarkers in large sample populations TSQs High-End Budget Limited 5

6 Orbitrap Fusion Tribrid Mass Spectrometer Unmatched Analytical Performance Revolutionary performance Unprecedented usability Exceptional versatility Orbitrap Fusion Tribrid: (trī-brǝd), n. three Mass Analyzers working in parallel to produce unmatched analytical results 6

7 Orbitrap Fusion Tribrid Mass Spectrometer Dual-pressure linear ion trap Ultra-high-field Orbitrap mass analyzer Ion-routing multipole Quadrupole mass filter Active beam guide (ABG) EASY-ETD (and IC) ion source 7

8 Flexible, Intuitive, Easy to Learn Software The Thermo Scientific Orbitrap Fusion Method Editor allows for drag and drop method building. Dynamic Scan Management for maximum data quality 8

9 Orbitrap Fusion Tribrid MS Unmatched Analytical Performance Scan rate FTMS 2 15 Hz Scan rate ITMS 2 Hz Max resolution 45K at Quad isolation down to.4 amu Ion trap isolation down to.2 amu Mass Accuracy 1 ppm Dissociation CID, HCD, ETD MSn Up to MS 1 in ion trap or Orbitrap Analyzers Q, FTMS, ITMS Detectors Ion Trap, Orbitrap Compact 1186 x 674 x 65 mm (w, d, h) 45, Resolution to remove spectral interferences CID/HCD/ETD detected by the Ion Trap or Orbitrap at any level of MS n for maximum experimental flexibility Powered by the new Dynamic Scan Management architecture that lets every user easily get the maximum information from every sample 9

10 Mass Accuracy Matters! Accurate mass improves the ability to confidently identify unknown compounds ppm error = (mass error / MW) x 1 6 EXAMPLE: Reserpine (C 33 H 4 N 2 O 9 ) has a protonated ion at Mass Error (ppm) # of Possible Chemical Formulae (using only C, H, N & O) 165 (.1 daltons) (.3 daltons) 1 1

11 Orbitrap Fusion Tribrid Mass Spectrometer Challenge Generate absolutely most accurate data to facilitate compound identification. Orbitrap Fusion MS EASY IC (internal calibration): new ETD source allows use of internal calibration for sub-ppm mass accuracies. On/off toggle in SW. No ion suppression by IC. 11

12 Reagent/Internal Calibrant Source Tube lens is replaced with discharge ion source for IC & ETD 12

13 Internal Calibration: LC/MS of Omeprazole Metabolites 13

14 Internal Calibration: LC/MS of Omeprazole Metabolites H+ 1 O N N H S O N O = O R e l a t i v e I n t e n s i t y Time (min) 14

15 Internal Calibration: LC/MS of Omeprazole Metabolites.28 ppm.55 ppm.47 ppm.55 ppm.55 ppm.47 ppm.35 ppm.55 ppm.36 ppm.28 ppm.47 ppm.55 ppm.28 ppm.3 ppm.2 ppm.19 ppm.19 ppm -.6 ppm.19 ppm.55 ppm.11 ppm.19 ppm -.14 ppm.19 ppm Time (min) 15

16 Internal Calibration: LC/MS of Omeprazole Metabolites.28 ppm -.5 ppm.11 ppm.55 ppm.47 ppm.55 ppm.55 ppm.47 ppm.11 ppm.35 ppm.3 ppm.28 ppm.55 ppm.36 ppm.47 ppm -.14 ppm.3.28 ppm ppm -.55 ppm -.22 ppm.28 ppm.3 ppm -.8 ppm.19 ppm.28 ppm.3 ppm.97 ppm.19 ppm -.6 ppm ppm Time (min).55 ppm.47 ppm.2 ppm.19 ppm.55 ppm.19 ppm.19 ppm -.14 ppm.19 ppm Time (min) 16

17 Easy-IC Providing Sub-ppm Mass Accuracy in MS and MS/MS Orbitrap Fusion MS for FS, 15k for HCD MS2, with UHPLC. Sample, Urine. Analyte: L-Tryptophan, C 11 H 12 N 2 O 2, [M+H] + = With IC Avg =.4 ppm Without IC Avg = 1.5 ppm MS Time (min) ASMS 13 poster MP45 by Junhua Wang et al With IC 1.4 ppm H 2N HCD MS/MS HN HN 5.2 ppm HN O 4.8 ppm -.1 ppm OH 3 HN HN N HN H Without IC 2.9 ppm ppm 1.7 ppm 1.9ppm ppm 2. ppm 2.1 ppm ppm 1.1 ppm HO NH.9 ppm 1.8 ppm O 1.1 ppm 17

18 Orbitrap Fusion Tribrid Mass Spectrometer Challenge Separation of distinct isobars in complex mixtures Orbitrap Fusion MS Orbitrap Fusion can achieve up to 45, resolution. 18

19 See the Fine Isotope Pattern with Ultra-high Resolution L-Methionine C 5 H 11 NO 2 S PositiveMode #656 RT: 2.64 AV: 1 NL: 1.48E8 T: FTMS + p ESI Full ms [ ] R= ppm Positive Observed R= R= R=3k R=6k R=1k R=24k R=45k C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:4) Chrg R= ppm Positive Simulated R=45, R= R= C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:4) Chrg C 4 H 1 18 O 2 33 S C 5 H 12 O 2 N 33 S C 4 13 CH 12 O 2 NS C 5 H 12 O 17 ONS C 13 5 C 14 CH 18 9 O 17 O ASMS 13 poster MP45 by Junhua Wang et al. 19

20 Resolving the Most Complex Sample Nano-infusion of NIST SRM195 plasma extract, Negative mode R= or ? D-Glucose or Paraxanthine? R= R= R= R= R=51372 R= D-Glucose C6H12O6 Paraxanthine C7H8N4O R=74 R=6, R=2875 R=24, R=14766 R=1, R=51682 R=45, D-Glucose R= ppm.55 ppm Paraxanthine Confirmed with STD mixture R=6, R= R= R= R= R=6786 R=24,.56 ppm R= ppm.55 ppm R=1, 1.7 ppm R=45,.55 ppm R= ppm.55 ppm ASMS 13 poster MP45 by Junhua Wang et al.

21 Orbitrap Fusion Tribrid Mass Spectrometer Challenge Achieve maximum coverage of the metabolome/lipidome. Many molecules require special fragmentation conditions to reveal their structural information. Orbitrap Fusion MS Instrument architecture allows for extremely fast MS/MS and full flexibility of MS n experiments for structural analysis including novel workflows such as HCD 3 and MS 2 HCD followed by MS 3 CID. 21

22 Method 1: The Super Q Exactive MS Mode MS in Orbitrap Data dependent HCD MS/MS in Orbitrap MS OT, R=7K Full MS.5s HCD MS 2 OT, R=17.5K MS 2 Top1 MS 2 Top2 RT: SM: 5G Time (min) MS OT, R=6K Full MS MS 2 Top1 MS 2 Top2 MS 2 Top3 MS 2 Top4 MS 2 Top5 22

23 .5s RT: Time (min) NL: 1.68E8 Base Peak F: ms MS E3_FS6k_Uri ne_hcdit_su perit5ms NL: 3.44E6 Base Peak F: ms2 MS E3_FS6k_Uri ne_hcdit_su perit5ms MS Orbitrap, R=24K Full MS MS 2 Top1 MS 2 Top2 MS 2 Top3 MS 2 Top4 MS 2 Top5 23

24 Method 3: MS n Enables de novo Compound ID Standards Standards Infusion (or LC) Add HR/AM MS n Library Match Score Above Threshold IDs MS n technology Spectral library technology Algorithms for structuralspectral investigation Unknowns LC-MS n Tree Search Match Score Below Threshold Individual Spectrum Search Partial Structures Structure Candidates Sheldon, Mistrik, Croley, J Am Soc Mass Spectrom, 9, :

25 Method 3: HR/AM Ion Trees for Unknown Identification Full MS in Orbitrap Data dependent HCD MS 2 in ion trap Data dependent CID MS 3 in Orbitrap.5-.8s MS CID MS 3 OT.6 sec HCD MS 2 IT Time (min) Full MS HCD/CID MS 2 in IT/OT MS 2 Top1 MS 2 Top2 HCD/CID MS 3 in IT/OT MS 2 Top1 MS 2 Top2 MS 2 Top3 MS 2 Top4 De novo structural elucidation: capture substructure with MS n ion tree 25

26 HCD MS n is Unique to Orbitrap Fusion MS OTMS> HCD OTMS 2 > HCD OTMS 3 HCD FTMS2 Scan Full FTMS Scan Orbitrap LPC HCD FTMS3 Scan HPC Ion Trap MP Q1 C-Trap Ion Routing Multipole Full Orbitrap MS scan Parent ion isolation in Q1 and HCD MS 2 > OTMS detection of MS 2 fragments HCD fragment isolation in the ion trap and HCD MS 3 Orbitrap MS detection of HCD MS 3 fragments 26

27 Structural Homology Identified by MS n Ion Tree Search 1 MS sec NL: 3.38E6 Base Peak = MS b667jwurine_hcdit_ci DOTtop2_5ms Time (min) CID MS 3 OT HCD MS 2 IT CID MS 3 OT HCD MS 2 IT O HN N NH isomers NL: 1.59E5 Base Peak F: ms2 MS b667jwurine_hcdit_ci DOTtop2_5ms NL: 2.93E4 Base Peak F: ms3 MS b667jwurine_hcdit_ci DOTtop2_5ms Theophylline MS 3 Library Match score Query 1.8 ppm 1.9 ppm 27

28 The Industry s Leading Portfolio of MS Solutions Exactive Series MS Non-targeted Analysis HR/AM Tribrid Orbitrap MS Metabolomics Proteomics Bioanalysis Quantitative Food Safety Environmental Clinical/Toxicology Applied Markets Triple Quads Transform Your Science MS, MS n Research Markets Ion Traps Biomarker Discovery Proteomics Metabolism Qualitative Metabolomics PTM Analysis Lipidomics Targeted Analysis 28

29 29 Transform Your Science