1. Metabolomics MS and Data Analysis PCB 5530 Tom Niehaus Fall 2015

2. Learning Outcomes - Learn the basics of Mass Spectroscopy - Mention statistical methods of data analysis - Go over example metabolomics datasets - Search for an unknown compound

3. Detection Mass Spectrometry mass spectrometry is a technique to measure the mass of ions (m/z) All mass spectrometers perform three main tasks: 1) Ionize molecules: 2) Use electric and magnetic fields to accelerate ions and manipulate their flight: 3) Detect ions (convert to electronic signal):

4. Detection Mass Spectrometry Example mass spectrum: m/z relative abundance

5. Detection Mass Spectrometry Time [min] Normalized Intensity or TIC m/z Normalized Intensity Chromatogram (GC-MS) 166 97 129 83 61 47 35 119 11270 Mass spectrum Peak selector

6. 6 Adduct formation – expect the unexpected Statistics: Adducts in NIST12 MS/MS DB (80,000 spectra) Most common adducts for LC-MS ([M+H]+ [M+Na]+ [M+NH4]+ [M+acetate]+) …around 290 different adducts

7. There are several types of mass spectrometers: - TOF (time of flight)- Q, QQQ (quadrupole) - Ion Trap- Orbitrap - FTICR (Fourier transform ion cyclotron resonance) Mass Spectrometry Mass Spectrometers QuadTOF

8. Mass Spectrometry Definitions and concepts Resolution (resolving power) Accuracy Mass range RP(FWHM) = measured mass / peak width at 50% peak intensity Difference in true mass and measured mass Range of ions that can be detected (typically 50-1000 m/z)

9. Mass Spectrometry Why is resolution important? High resolution is needed to determine the accurate mass High resolution is also needed to determine accurate isotopic patterns Note: -monoisotopic vs ave mass

10. Data Analysis Goals Identify all peaks In practice this is very difficult if not impossible quantification or semi-quantification of compounds Standards are needed to quantify a metabolite. For rigorous quantification, samples must be spiked with an isotopically labeled standard. Various statistical tests to look for differences in the treatment groups e.g. PCA, MCA, ANOVA Huge data files Generally relative abundance of metabolites (e.g. –fold change in KO vs WT)

11. Data Analysis Identifying peaks MS libraries can identify peaks (mostly GC/MS), especially when combined with RT information (GC/MS only): e.g. NIST library

12. Data Analysis Example PCA analysis Figure 2: Multivariate statistical analysis results of serum metabolites in the pCPA-treated mice (n = 40) and the control mice (n = 40). Metabolomics Approach Reveals Integrated Metabolic Network Associated with Serotonin Deficiency (PMID: 26154191)

13. Data Analysis Identifying peaks -Fold changes in metabolites can be visualized by a heat map. PMID: 26154191 Figure 3: Heat map denoting fold changes (over normalized means) of the 21 biomarkers in mice injected with increasing dosages of pCPA and the control mice (n = 10).

14. Data Analysis Example output of a metabolomics experiment Open GC-TOF-MS dataset from class website: -How many compounds identified? How many significant -fold changes -Pathway analysis at http://www.metaboanalyst.ca/MetaboAnalyst/http://www.metaboanalyst.ca/MetaboAnalyst/ -enter compound names or KEGG IDs for significant -fold changes -choose organism ‘E. coli’ and submit -Which pathways are affected in this dataset? Open HILIC-TOF-MS dataset from class website: -How many compounds identified? How many significant -fold changes -How many unidentified peaks? -Can you identify an unknown peak with a significant fold change

15. Data Analysis Activity 1: Identifying peaks Can you find sucrose in a MS dataset? Example: sucrose (C 12 H 22 O 11 )

16. Data Analysis Activity 1: Identifying peaks Accurate mass can help determine the chemical formula: Example: sucrose (C 12 H 22 O 11 ) -Determine monoisotopic mass at http://www.chemspider.com/http://www.chemspider.com/ (342.116211 Da) -Determine M+H from MS adduct excel sheet (class website) (343.123487 Da) Lets say you find that mass in the dataset, but is it really sucrose? -Download Molecular weight calculator at http://www.alchemistmatt.com/mwtwin.html http://www.alchemistmatt.com/mwtwin.html -Open formula finder under tools -enter molecular weight target: 342.116211 -how many isobars are at 2 ppm? 0.1 ppm -enter 342.116211 at chemspider, how many isomers?