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Data Acquisition and Analysis in Mass Spectrometry Based Metabolomics

Data Acquisition and Analysis in Mass Spectrometry Based Metabolomics. Pavel Aronov BioCyc workshop October 27, 2010. Outline. Fundamentals of Mass Spectrometry Data Acquisition and Analysis in GC-MS based Metabolomics Data Acquisition and Analysis in LC-MS based Metabolomics.

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Data Acquisition and Analysis in Mass Spectrometry Based Metabolomics

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  1. Data Acquisition and Analysis in Mass Spectrometry Based Metabolomics Pavel Aronov BioCyc workshop October 27, 2010

  2. Outline • Fundamentals of Mass Spectrometry • Data Acquisition and Analysis in GC-MS based Metabolomics • Data Acquisition and Analysis in LC-MS based Metabolomics

  3. How to analyze tryptophan or any other metabolite? Two most common techniques in analytical chemistry to determine or confirm chemical structure: Nuclear Magnetic Resonance/NMR (1940s, Felix Bloch at Stanford University) Excellent structural information Mass Spectrometry (1900s, JJ Thompson at Cambridge University) Excellent sensitivity

  4. What is a mass spectrometer? Atmosphere Mass Spectrometer Vacuum M Ion Source Mass Analyzer Detector M+ M+ M M M+ M+ Measured value: mass-to-charge ratio M/Z

  5. Mass Units • Unit of mass: 1/12 mass of carbon-12 atom 1 u or 1 Da • Unit of mass-to-charge 1 Da / z = 1 Th (Thompson) m/z 205 For metabolites usually z = 1, Hence 1 Da is equivalent to 1 Th

  6. Monoisotopic vs Average Mass Two stable isotopes important in biochemistry Carbon-12 (100 %) and Carbon-13 (~1.1 %) Sulfur-32 (100 %) and Sulfur-34 (4.4 %) Tryptophan statistically can contain: no carbon-12 (M): 204.09 Da (100 %) one carbon-13 (M+1): 205.09 Da (11.9 %) two carbons-13 (M+2): 206.09 Da (1.4 %) These are monoisotopic masses Average mass = (204.09 *100 + 205.09*11.9 + 206.09*1.4)/113.2 = 204.22 (molecular weight, g/mol)

  7. Mass defect 1H (p+e-) 1.0078 u 12C 12.0000 u 14N 14.0031 u 16O 15.9949 u n 1.0087 u Carbon-12: 6 protons, 6 neutron and 6 electrons 6 x 1.0078 u + 6 x 1.0087 u = 12.0990 u Mass Defect = 12.0990 u – 12.0000 u = 0.0990 u E = mc2 0.1 u = 93 MeV 

  8. Elemental composition from accurate mass 1H 1.0078 u 12C 12.0000 u 14N 14.0031 u 16O 15.9949 u What is 28 u? N2 (2 x 14 u), CO (12 u + 16 u) or C2H4 (2 x 12 u + 4 x 1 u)? What is 28.0313 u? [high accuracy] C2H4 (2 x 12.0000 u + 4 x 1.0078 u)

  9. High resolution mass spectrometry 562.19 100 561.18 % 563.20 564.20 0 561.14 100 562.10 % 563.06 0 m/z 560 561 562 563 564 High Resolution: R = 561/0.06 ~ 9,000 TOF: 7,000-50,000 Orbitrap: 104-105 FT ICR: 105-106 Nominal Mass Resolution (<1000) R = 561/0.8 ~ 700 Quadrupoles and ion traps, some TOFs 0.06 amu FWHM 0.8 amu FWHM 9 9

  10. Mass of an electron becomes important at high accuracies Two types of ions in mass spectrometry: Odd Electron (OE) Ions Typically generated by electron ionization (GC/MS): Even Electron (EE) Ions Typically generated by chemical ionization techniques and electrospray e 0.00055 Da 204.08988 Da (2.6 ppm error) 204.08933 Da (true mass) 205. 09715 Da (true mass) 205. 09770 Da (2.6 ppm error) Modern instruments can achieve < 1 ppm accuracy

  11. Identification based on accurate mass Acquired spectrum Matching accurate mass and isotopic peak ratio Theoretical spectrum Error = -0.00013 Da/212.0023 Da * 1000,000 = 0.6 parts per million (ppm)

  12. Confirmation of structure from isotopes (M+2) Acquired spectrum Matching accurate mass and isotopic peak ratio Theoretical spectrum

  13. Tandem Mass Spectrometry Atmosphere Mass Spectrometer Vacuum M Ion Source Mass Analyzer 1 Mass Analyzer 2 Detector M+ F+ M+ F+ Collision Cell M M M+ F+ M+ F+ HPLC

  14. MS/MS of isomers Prostaglandin A1 336.2301 amu Prostaglandin B1 336.2301 amu

  15. Chromatography ) 14.40 100 16.73 11.82 18.84 10.43 9.00 % 30.05 20.77 28.44 22.53 27.07 24.16 25.66 0 Time 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 Separation by volatility and polarity (gas chromatography/GC) or polarity (liquid chromatography/LC) C12 C14 C10 C16 C9 Gas chromatography of hydrocarbons C8 C18 C30 C20 C22

  16. 2D dimensionality of metabolomics datain LC-MS and GC-MS

  17. GC-MS and LC-MS GC LC -Derivatization usually required (except VOC) -Upper mass limit at ~400-500 amu -Preferred for small polar metabolites (primary metabolism) -Relatively high peak capacity -No derivatization usually required -Upper mass is limited by column permeability -Preferred for bigger molecules (e.g. some lipids, secondary metabolites) -Relatively low peak capacity -EI ion source (extensive fragmentation, reproducible, libraries available -CI ion source (little fragmentation, advantage for accurate mass measurement -ESI ion source (ionic compounds, ion suppression) -APCI ion source (less ion suppression and more amenable for non polar compounds than ESI but usually lower sensitivity) MS

  18. Types of Experiments in Metabolomics targeted non-targeted • Number of analyzed metabolites is limited by the number of available standards • Absolutequantitation of metabolites (nM, mg/mL) • Selective MS detectors (quadrupoles, triple quadrupoles) • Number of analyzed metabolites is limited by capacity of analytical instrumentation • Relativequantitation of metabolites (fold) • Scanning MS detectors (ion trap, TOF, FT)

  19. Bottlenecks in Metabolomics ASMS09 survey: metabolomics bottlenecks 9-Other; 2% 8-Data acquisition/throughput; 3% 7-Validation/Utility Studies; 5% 6-Statistical analysis; 5% 1-Identification of metabolites; 35% 5-No opinion; 6% 4-Sample preparation; 8% 3-Data processing/reduction; 14% 2-Assigning biological significance; 22% throughput (3 %) vs. post-acquisition bottlenecks (5 + 35 + 22 + 14 = 76 %)

  20. GC-MS based metabolomics: overview 50 - 600 (400) amu mass range mono- and disaccharides, amino acids, fatty acids (mostly primary metabolites) Derivatization usually required

  21. GC-MS: derivatization 40 mg/mL in pyridine at 37˚C for 90 min • Prevents α-ketoacids from thermal decarboxylation • Keeps sugars in open conformation to minimize number of conformation and relieve steric hindrences for next step α/β epimers Syn/anti isomers

  22. GC-MS: derivatization MSTFA, 1% TMCS at 50˚C for 30 min • Substitution of active hydrogens • Incomplete derivatization possible

  23. GC-MS data analysis

  24. Electron Ionization in GC-MS 70 eV >> energy of chemical bond • Highly reproducible • Extensive fragmentation • Often no molecular ion observed EI: alpha-cleavage [a ] more common CID MS/MS: inductive cleavage [i ] common

  25. GC-MS: present and future • Current GC-MS metabolomics platforms use: • 1) nominal resolution mass analyzers • (no accurate mass and elemental composition) • 2) electron ionization ion source • OE molecular ions, extensive fragmentation, • often molecular ion is not observed • Advantages: • Low cost • Good chromatographic separation for many small polar • metabolites after derivatization • 3) Extensive libraries of fragmentation spectra help identification • 4) Retention time is to some extent predictable (retention indices) • Trends: • 1) Development of high resolution instruments for GC/MS • 2) Development of soft ionization sources similar to LC/MS • (EE ions, no fragments)

  26. GC-MS data analysis • Deconvolution of mass spectra based on chromatographic profiles (e.g freeware AMDIS) • Identification of metabolites based on matching to spectral libraries and retention indices • Automated processing routines exist for some GC-MS instrument (SetupX and BinBase)

  27. Application Examples - cells Glycine-2TMS + cells

  28. Application Examples: AMDIS Peak of interest Acquired mass spectrum Library mass spectrum (glycine-2TMS)

  29. LC-MS based metabolomics Combination of ionization modes is preferred (ESI, APCI, +, -) Reversed phase LC for non-polar metabolites and hydrophilic interaction chromatography (HILIC) for polar metabolites Detection of spectral “features” (ions) using metabolomics software Identification based on accurate mass, and fragmentation (MS/MS libraries)

  30. Electrospray Ionization (ESI) Positive ESI R + H+ R – H+ [R+H]+ [R – H]+ Negative ESI

  31. Combination of Acquisition Modes Separation modes: Reversed phase and HILIC Ionization modes: ESI and APCI or combined ESI/APCI (MM) Ionization polarities: + and - Nordstrom A. et al, Anal Chem, 2008.

  32. RP and HILIC liquid chromatography Creatinine Reversed Phase C18 Creatinine Aminopropyl HILIC Better retention for polar molecules

  33. LC-MS: Data Analysis • Alignment of chromatograms (optional) • Detection of ‘features’ in mass chromatograms • Removal of isotopic peaks, adducts, fragments etc to improve statistics • Statistical analysis • Identification based on accurate mass, MS/MS spectra and comparison with standards

  34. Example: Search for bacterial metabolites in humans comparing two groups: controls and people who underwent colectomy (no colon bacteria) Initially software detected 900 features in positive ESI mode After features with missing chromatographic profile were removed 769 features left (visual inspection) After isotopes were removed, 554 features left. Only at this point, these are likely molecular ions of individual metabolites

  35. Adducts M + H M + NH4 M + Na M + NH4 M + H M + Na

  36. Fragments in LC-MS Hyppuric acid m/z 118.0651 Hyppuric acid C8H8N – indole? No, fragment of hyppuric acid Not confirmed by GC-MS either

  37. Identification tools Accurate mass search (BioCyc, HMDB, Metlin) MS/MS search (Metlin, MassBank) In addition, many MS manufacturers offer proprietary tools for structure elucidation

  38. MassBank MS/MS sulfate m/z 132 C8H6NO

  39. LC-MS Data Analysis Summary • Not every peak detected by a mass spectrometer represents an individual metabolite • Automated data processing helps to reduce the amount of routine work, however human intervention is still required • Accurate mass measurements and MS/MS allow to determine elemental composition of unknowns and their structural components. Confirmation with chemical standards is still required

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