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
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
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
Orbitrap Delivers the Highest Quality Data Better data, Better results! Highest Resolution Best Mass Accuracy Widest Dynamic Range Excellent Sensitivity 4
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
Orbitrap Fusion Tribrid Mass Spectrometer Unmatched Analytical Performance Revolutionary performance Unprecedented usability Exceptional versatility Orbitrap Fusion 2 3 1 Tribrid: (trī-brǝd), n. three Mass Analyzers working in parallel to produce unmatched analytical results 6
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
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
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
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 69.2866 Mass Error (ppm) # of Possible Chemical Formulae (using only C, H, N & O) 165 (.1 daltons) 9 1 13 5 7 3 4 2 2 1 2.5 (.3 daltons) 1 1
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
Reagent/Internal Calibrant Source Tube lens is replaced with discharge ion source for IC & ETD 12
Internal Calibration: LC/MS of Omeprazole Metabolites 13
Internal Calibration: LC/MS of Omeprazole Metabolites H+ 1 O N N H S O N 362.1169 O = O R e l a t i v e I n t e n s i t y 1 2 3 4 5 6 7 8 9 Time (min) 14
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 3.7 3.8 3.9 4. 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Time (min) 15
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 3.7 3.75 3.8.11 ppm Time (min).55 ppm.47 ppm.2 ppm.19 ppm.55 ppm.19 ppm.19 ppm -.14 ppm.19 ppm 3.7 3.8 3.9 4. 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Time (min) 16
Easy-IC Providing Sub-ppm Mass Accuracy in MS and MS/MS 1 9 8 7 6 5 4 3 1 1 9 8 7 6 5 4 3 1 Orbitrap Fusion MS operating @1K for FS, 15k for HCD MS2, with UHPLC. Sample, Urine. Analyte: L-Tryptophan, C 11 H 12 N 2 O 2, [M+H] + =5.9715 With IC Avg =.4 ppm 5.9717 5.9721 5.9721 5.9724 5.9723 5.9717 5.9717 5.9715 5.9718 5.97 Without IC Avg = 1.5 ppm 5.9741 MS 5.9747 5.9749 5.9746 5.974 5.977 5.9746 5.9772 5.9735 5.9738 2.76 2.77 2.78 2.79 2.8 2.81 2.82 2.83 2.84 2.85 2.86 Time (min) ASMS 13 poster MP45 by Junhua Wang et al. 1 9 8 7 6 1 9 8 7 6 5 4 3 1 With IC 1.4 ppm H 2N HCD MS/MS HN 118.653 HN 5.2 ppm HN O 4.8 ppm -.1 ppm OH 3 HN HN 132.89 N HN H 2 17.615 188.763 1 91.5422 8 1 1 14 16 18 Without IC 2.9 ppm 118.6544.4 ppm 1.7 ppm 1.9ppm 146.6.7 ppm 2. ppm 2.1 ppm 146.633 1.4 ppm 1.1 ppm 132.815 188.779 17.63 8 1 1 14 16 18 HO NH.9 ppm 1.8 ppm O 1.1 ppm 17
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
See the Fine Isotope Pattern with Ultra-high Resolution L-Methionine C 5 H 11 NO 2 S PositiveMode1351892831 #656 RT: 2.64 AV: 1 NL: 1.48E8 T: FTMS + p ESI Full ms [1.-212.8] 15.584 R=6276 1 9 8 7 6 5 4 3 1.7ppm Positive Observed 151.618 R=62152 152.542 R=6182 15. 15.5 151. 151.5 152. 1 8 6 4 151.5 151.6 R=3k R=6k R=1k R=24k R=45k C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:4) Chrg 1... 1 9 8 7 6 5 4 3 1 15.583 R=499553.ppm Positive Simulated R=45, 151.617 R=499368 152.541 R=499443 15. 15.5 151. 151.5 152. C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:4) Chrg 1... 1 8 6 4 151.554 14 C 4 H 1 18 O 2 33 S 151.577 C 5 H 12 O 2 N 33 S 151.617 C 4 13 CH 12 O 2 NS 151.625 C 5 H 12 O 17 ONS 151.646 C 13 5 C 14 CH 18 9 O 17 O 151.5 151.55 151.6 151.65 ASMS 13 poster MP45 by Junhua Wang et al. 19
Resolving the Most Complex Sample Nano-infusion of NIST SRM195 plasma extract, Negative mode 8 6 4 1 167.213 R=35365 179.561 or 179.575? D-Glucose or Paraxanthine? 172.99141 R=49792 18.6662 R=56177 187.713 R=33315 215.3278 R=35614 3.49 21.84235 R=51372 R=53512 16 165 17 175 18 185 19 195 5 21 215 2 D-Glucose C6H12O6 Paraxanthine C7H8N4O2 1 9 8 7 6 5 4 3 1 1 9 8 7 6 5 4 3 1 179.5747 R=74 R=6, 179.55 179.6 179.5759 R=2875 R=24, 179.55 179.6 1 9 8 7 6 5 4 3 1 1 9 8 7 6 5 4 3 1 179.5763 R=14766 R=1, 179.55 179.6 179.5743 R=51682 R=45, D-Glucose 179.569 R=5377.55 ppm.55 ppm Paraxanthine 179.55 179.6 Confirmed with STD mixture. 1 9 8 7 6 5 4 3 1 9 8 7 6 5 4 3 1 R=6, 179.573 R=6766 1 9 8 7 6 5 4 3 1 179.558 R=13 179.574 R=1484 179.5 179.55 179.574 179.6 179.65 179.5 179.55 179.6 179.574 179.65 1 R=3464 1 R=6786 R=24,.56 ppm 179.56 R=275 1.1 ppm.55 ppm 9 8 7 6 5 4 3 1 R=1, 1.7 ppm R=45,.55 ppm 179.56 R=56282.55 ppm.55 ppm 179.5 179.55 179.6 179.65 179.5 179.55 179.6 179.65 ASMS 13 poster MP45 by Junhua Wang et al.
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
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: 1.6345-1.8627 SM: 5G 1 9 8 7 6 5 4 3 1 1 9 8 7 6 5 4 3 1 1.698 1.7699 1.7789 1.7879 1.7969 1.859 1.8149 1.716 1.6798 1.8241 1.7251 1.761 1.677 1.6525 1.75 1.8333 1.8516 1.7298 1.6572 1.6846 1.7118 1.741 1.6677 1.7657 1.7927 1.817 1.8393 1.64 1.66 1.68 1.7 1.72 1.74 1.76 1.78 1.8 1.82 1.84 1 Time (min) MS OT, R=6K Full MS MS 2 Top1 MS 2 Top2 MS 2 Top3 MS 2 Top4 MS 2 Top5 22
.5s RT: 2.5298-2.5478 1.4 1.2 1..8.6.4.2. 1.4 1.2 1..8.6.4.2 2.5384 2.5465 2.5315 2.535 2.5394 2.5432 2.5371 2.5445. 2.53 2.535 2.54 2.545 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
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, : 37-376 24
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 1 1 1 MS 3.1791 3.1891 CID MS 3 OT.6 sec HCD MS 2 IT 3.178 3.18 3.182 3.184 3.186 3.188 3.19 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
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
Structural Homology Identified by MS n Ion Tree Search 1 MS 3.1791 3.1891.6 sec NL: 3.38E6 Base Peak = 181.73-181.739 MS b667jwurine_hcdit_ci DOTtop2_5ms 1 1 1 8 6 4 3.178 3.18 3.182 3.184 3.186 3.188 3.19 Time (min) 1 8 6 4 CID MS 3 OT HCD MS 2 IT 69.1 6 8 1 1 14 16 18 CID MS 3 OT 69.447 95.8 96.556 15.1 6 8 1 1 HCD MS 2 IT O HN 124.2 N NH 1 8 6 4 141.91 69.1 isomers 95.773 15.77 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 124.153 18.96 141.9145 18.9644 6 8 1 1 14 16 18 Theophylline MS 3 Library Match score 936.2 Query 1.8 ppm 1.9 ppm 27
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