A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzcloud Library Junhua Wang, 1 David A. Peake, 1 Robert Mistrik, 2 Yingying Huang 1 1 Thermo Fisher Scientific Inc, San Jose, CA, USA; 2 HighChem, Ltd., Bratislava, Slovakia
Overview Purpose: To demonstrate the capability of the Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometer in combination with MS n library for metabolite structural identification. Methods: UHPLC coupled to the Orbitrap Fusion MS to simultaneously perform global profiling and structural elucidation with MS n. Results: Within UHPLC timescale (2-3 second FWHM), >1 cycles can be finished with fast ion trap HCD MS 2 for top 11 precursors, each with ion trees of MS 2 -MS 3 (top2)-ms 4 (top2). Introduction The structural identification of metabolites represents a significant challenge in metabolomics study. Multistage mass spectrometry (MS n ) is a powerful tool for compound identification and structural elucidation that goes beyond identifying the exact same compounds, to discovering additional compounds with structural homology to those in the library. Combining with high resolution accurate mass (HRAM) measurement, MS n is highly e ective in identifying the unknown but biologically relevant compounds in metabolomics studies[1]. However, the speed on current platforms is yet to be e ectively compatible with UHPLC front end, which limited such application in biological samples. Presented here is a new platform to identify endogenous metabolites using a novel high performance Orbitrap hybrid instrument in conjunction with UHPLC and an MS n library. Methods Sample Preparation Urine samples from an adult male were analyzed. The urine samples were diluted 1 to 1 with water and processed with a 3kD MWCO filter. For plasma samples, proteins were removed with cold methanol and centrifugation, followed by centrifugal evaporation at 35 o C. The residue was reconstituted in MeOH/Water, 1/9, and also processed with a 3kD MWCO filter. Liquid Chromatography UHPLC separation was implemented on a Dionex Ultimate 3 HPG (highpressure gradient) pump using Hypersil GOLD RP C18 column at 45 L/min, column temperature at 55 o C. LC solvents were.1% FA (A) and.1% FA in MeOH (B). A linear gradient was applied from.5-5% B for 5.5 min, followed by increasing to 98% at 6 min, held 98% B for 6 min, then decreased to.5% at 13min, and equilibrated for another 2 min. FIGURE 1. Schematic of Orbitrap Fusion Tribrid Mass Spectrometer Dual-pressure linear ion trap Large- surface- Ion-routing Area detector multipole (LSAD) Ultra-high-field Orbitrap mass analyzer C-Trap Active beam guide (ABG) Quadrupole mass filter EASY-Internal Calibration ion source S-Lens 2 A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzcloud Library
An Improved Metabolomics Workflow A traditional workflow for metabolite profiling and identification is based on a full scan LC/MS experiment followed by targeted MS/MS confirmation. The major problems associated with this methodology are: 1) Instrumentation limitations Does not allow simultaneous profiling and MS n in a single run, the scan speed su ers. Identification is usually based on accurate mass and MS 2, but a large number of LC peaks remain not identifiable. 2) MS/MS library limitations MS/MS spectral search is usually manually performed and timeconsuming; Automated scoring for spectral matching is usually not available; The fragmentation needs to be manually interpreted, which is not only time-consuming but also prone to errors. 3) There is no good solution for unknowns. The Orbitrap Fusion MS enables fast parallel data acquisition on a UHPLC timescale with a variety of dissociation choices The Orbitrap Fusion MS o ers ultimate flexibility in fragmentation methods at all stages of MS n analysis, with fragment ions detected by either mass analyzer. CID-HCD, HCD-CID, and HCD-HCD can all be set up as simple as the conventional CID-CID. This complete flexibility facilitates the examination of fragmentation pathways that leads to the most comprehensive structural information. mzcloud MS n library enables MS n search for both high and low resolution mzcloud is a multistage MS library. Based on the spectral tree similarity, an algorithm called precursor ion fingerprinting (PIF) enables the identification of compounds even if they are not in the library, and the fragmental peaks can be annotated [2]. Powered by the novel MS and the mzcloud library, an improved, more connected workflow is shown below: Major benefits of the improved workflow: 1) HCD MS/MS can be performed simultaneously with full scan MS on the UHPLC timescale with ion trap detection at >22 Hz. 2) Simultaneous ddms n ion tree with flexible combinations of CID and/or HCD fragmentation with ion trap and/or Orbitrap TM detection. 2) Both the MS 2 and MS n will be automatically searched in mzcloud, the matching entry will be scored based on PIF, and the fragments will be annotated. 4) De novo structural identification is enabled by using this workflow. Thermo Scientific Poster Note PN ASMS13_MP 45_JWang e 6/13S 3
Results FIGURE 1. The Orbitrap Fusion MS and the Drag and Drop Instrument Method Editor. Metabolite Identification Requires High Resolution and Isotope Ratio Measurements Structure elucidation of unknown small molecules by mass spectrometry is a great challenge. The first crucial step is to obtain correct elemental compositions. Natural occurring elements can be monoisotopic (F, Na, P, I) or polyisotopic (H, C, N, O, S, Cl, Br) [1]. The molecule L-Methionine C 5 H 11 NO 2 S contains multiple polyisotopic elements, especially the S, which result in multiple isotopic peaks needing resolving power above 24, at (FWHM). Figure 2A shows the better isotope pattern of molecular ion [M+1+H] +. With increasing resolution up to 45, (FWHM), all five isotope peaks are clearly resolved. Figure 2B shows the simulated isotope pattern. FIGURE 2. Ultrahigh resolution resolves the isotopes A PositiveMode1351892831 #656 RT: 2.64 AV: 1 NL: 1.48E8 T: FTMS + p ESI Full ms [1.-212.8] 15.584 R=6276 1 B 9 8 7 6 5 4 3 1 L-Methionine C 5 H 11 NO 2 S.7ppm Positive Observed 151.618 R=62152 152.542 R=6182 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 Fine isotope pattern 151.5 151.6 C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:4) Chrg 1... R=3k R=6k R=1k! =24k R=5k 15.583 R=499553 1 9.ppm Positive 8 Simulated 7 R=5, 6 5 4 3 151.617 152.541 1 R=499368 R=499443 15. 15.5 151. 151.5 152. 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 4 A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzcloud Library
H O he Drag and Drop Instrument Metabolite Identification and Quantitation in Complex Samples Requires High Resolution D-Glucose C 6 H 12 O 6 and Paraxanthine C 7 H 8 N 4 O 2 co-exist in human blood at distinct concentration levels: 53 M (D-Glucose) vs. 1 M (Paraxanthine) [3]. Although D-Glucose is hundreds of fold more concentrated, it is less readily detectable because of its low ionization e ciency. Their masses di erentiate by.13 amu (179.561 vs. 179.574), making the detection of these individual components very challenging. Figure 3 shows that, with >3, resolving power, the M (D-Glucose) and 1 M (Paraxanthine) can be readily separated and quantified. FIGURE 3. Ultrahigh resolution resolves isobaric metabolites FIGURE 5. Rapid Ion Tree Acquis Urine Sample (1 MS, 2 MS 2 and 4 M (A) Ion trap HCD MS 2 followed by O precursor ion 181.721. (B) Spect MS 3 with MS 3 from theophylline with th must have the same substructure as th A.55 ppm 179.576 R=5542 1 9 8 7 6 5 4 3 1 Negative Full Scan 18.611 R=33 179 18 181 182 1 9 8 7 6 5 4 3 1 1 179.573 R=6766 179.5 179.55 179.6 179.65 9 8 7 6 5 4 3 1 R=6, R=24,.55 ppm 179.56 R=275 179.574 R=3464.56 ppm 1.1 ppm 179.5 179.55 179.6 179.65 1 9 8 7 6 5 4 3 1 179.558 R=13 179.574 R=1484 179.5 179.55 179.6 179.65 1 9 8 7 6 5 4 3 1 R=1, 1.7 ppm R=5, M 179.56 R=56282.56 ppm.55 ppm.55 ppm 179.574 R=6786 1 M 179.5 179.55 179.6 179.65 h Resolution and Isotope Ratio Metabolite Identification Requires High Accuracy of Mass Measurement in Both MS Full Scan Level and MS/MS Level lecules by mass spectrometry is a obtain correct elemental For an unknown metabolite, the number of calculated chemically possible formulae could be hundreds ( ppm tolerance window), but the possibilities significantly reduce to tens (3 ppm) and several if the tolerance window is narrowed to sub-ppm. B topic (F, Na, P, I) or polyisotopic (H, ionine C 5 H 11 NO 2 S contains multiple ich result in multiple isotopic peaks t (FWHM). Figure 2A shows r ion [M+1+H] +. With increasing isotope peaks are clearly resolved. ern. However, due to the large amount of known isomeric structures, one single formula may correspond to hundreds of structural isomers, thus structure identification of metabolite isomer using only MS full scan is not feasible. MS/ MS and MS n techniques can help solve this analytical problem. The Orbitrap Fusion MS has a built-in internal calibration functionality (Easy IC). Figure 4 shows LC-MS/MS data of L-Tryptophan from an urine sample with and without internal calibration. Sub-ppm mass accuracy was observed for MS and MS 2 fragments with IC activated. isomers Theophylline M Relative Abundance 1 9 8 7 6 5 4 3 1 1 FIGURE 4. Easy Internal Calibration 9 8 7 6 5 4 3 1 Full Scan MS with Int. Cal. Avg =.4 ppm 5.9717 5.9721 5.9721 5.9724 5.9723 5.9717 5.9717 5.9715 5.9718 5.97 5.974 5.9741 5.9747 5.9749 5.9746 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) without Int. Cal. Avg =1.5 ppm Relative Abundance Relative Abundance JWUrine_MS2_1k_6ms_bothLM #896 RT: 2.84 AV: 1 NL: 1.8E7 1 ppm T: FTMS + p ESI d Full ms2 5.1@hcd35. [5.-216.] HO 146.6 1 NH 9 1 ppm NL: 8 H 2 N.4 ppm 3.23E7 Base Peak 7 = 5.9525-.7 ppm 5.9935 F: ms MS 6 118.653.9 ppm JWUrine_FS24k _LM_Re 5.2 ppm O 4.8 ppm -.1 ppm OH 3 N 132.89 2 17.615 188.763 1 91.5422 NL: 2.85E7 8 1 1 14 16 18 Base Peak = PBUrine_MS2_1k_6ms_bothLM 5.9525- #824 RT: 2.82 AV: 1 NL: 8.68E6 5.9935 MS F: jwurine_fs24k_n FTMS + p ESI d Full ms2 5.9@hcd35. [5.-216.] 2.1 ppm olm_re 146.633 1 9 8 7 6 5 4 3 1 with Int. Cal. without Int. Cal. 2.9 ppm HCD MS/MS 118.6544 1.7 ppm 1.9ppm 2. ppm 1.1 ppm 1.8 ppm 132.815 17.63 188.779 1.1 ppm 8 1 1 14 16 18 Conclusion The Orbitrap Fusion MS o ers ultra hig ratio measurements of metabolites. This with a wide dynamic range be resol Calibration allows compound assignmen Orbitrap Fusion MS provides ultimate fle multiple structural pathways for more str The mzcloud library allows metabolite id an automated fashion. The identification and PIF algorithm opens the possibilit isomers. An improved metabolomics work References Score 1. Budzikiewicz H, Grigsby RD, Mass S 2. Sheldon MT, Mistrik R, Croley TR, J. 37-376 3. http://www.hmdb.ca/metabolites/h Qu Thermo Scientific Poster Note PN ASMS13_MP 45_JWang e 6/13S 5
FIGURE 5. Rapid Ion Tree Acquisition on UHPLC Timescale from a Urine Sample (1 MS, 2 MS 2 and 4 MS 3 in.6sec/cycle). (A) Ion trap HCD MS 2 followed by Orbitrap CID MS 3 were acquired for the precursor ion 181.721. (B) Spectral tree search in mzcloud matches the MS 3 with MS 3 from theophylline with the highest score, indicating the unknown must have the same substructure as theophylline. A 1 3.1791 Full Scan MS 3.1891.6 sec NL: 3.38E6 Base Peak = 181.73-181.739 MS b667jwurine_hcdit_ci DOTtop2_5ms 1 MS2 NL: 1.59E5 Base Peak F: ms2 MS b667jwurine_hcdit_ci DOTtop2_5ms 1 MS3 NL: 2.93E4 Base Peak F: ms3 MS b667jwurine_hcdit_ci DOTtop2_5ms 3.178 3.18 3.182 3.184 3.186 3.188 3.19 Time (min) 1 124.153 8 6 HCDIT-MS2 15.77 4 18.9644 141.9145 69.1 95.773 6 8 1 1 14 16 18 1 CIDOT-MS3 96.556 8 6 69.447 4 6 8 1 1 1 8 6 4 124.153 15.77 18.9644 141.9145 95.773 69.1 6 8 1 1 14 16 18 B Theophylline MS 3 isomers O N N N NH NH Score Query (down) and library spectra (up) match 1.5 ppm 1.5 ppm Conclusion The Orbitrap Fusion MS o ers ultra high resolution and high fidelity in isotope ratio measurements of metabolites. This allows metabolites in complex mixture with a wide dynamic range be resolved and detected. The Easy-Internal Calibration allows compound assignment be readily done with confidence. The Orbitrap Fusion MS provides ultimate flexibility that facilitates the examination of multiple structural pathways for more structural information at higher speed. The mzcloud library allows metabolite identification with MS 2 and/or MS n data in an automated fashion. The identification based on multistage MS spectral trees and PIF algorithm opens the possibility to identify unknown metabolites and isomers. An improved metabolomics workflow is enabled. References 1. Budzikiewicz H, Grigsby RD, Mass Spectrom. Rev. 6, 25(1):146-157. 2. Sheldon MT, Mistrik R, Croley TR, J. Am. Soc. Mass Spectrom. 9, (3): 37-376 3. http://www.hmdb.ca/metabolites/hmdb122 & HMDB186 6 A Platform to Identify Endogenous Metabolites Using a Novel High Performance Orbitrap MS and the mzcloud Library
www.thermoscientific.com 13 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientific, Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Thermo Fisher Scientific, San Jose, CA USA is ISO 91:8 Certified. Africa-Other +27 11 57 184 Australia +61 3 9757 43 Austria +43 1 333 5 34 Belgium +32 53 73 42 41 Canada +1 8 53 8447 China +86 1 8419 3588 Denmark +45 7 23 62 6 Europe-Other +43 1 333 5 34 Finland/Norway/Sweden +46 8 556 468 France +33 1 6 92 48 Germany +49 613 48 114 India +91 22 6742 9434 Italy +39 2 95 591 Japan +81 45 453 91 Latin America +1 561 688 87 Middle East +43 1 333 5 34 Netherlands +31 76 579 55 55 New Zealand +64 9 98 67 Russia/CIS +43 1 333 5 34 South Africa +27 11 57 184 Spain +34 914 845 965 Switzerland +41 61 716 77 UK +44 1442 233555 USA +1 8 532 4752 ASMS13_MP 45_JWang_E 6/13S