Metabolomics Part 2 Mass Spectrometry PCB 5530 Guillaume Beaudoin gbeaudoin@ufl.edu Fall 2016

Detection Mass Spectrometry Mass spectrometry (MS) separates ions in vacuum according to mass-to-charge ratio (m/z) All mass spectrometers must perform three main tasks: Ionize molecules Use electric and magnetic fields to accelerate ions and manipulate their flight in the gas phase Detect ions (convert to electronic signal)

Mass Spectrometry Sample Ion Source Mass Analyzer Detector GC LC GC LC Electron Ionization (EI) Chemical Ionization (CI) GC Electrospray Ionization (ESI) Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure Photoionization (APPI) LC Electron Ionization (EI) Chemical Ionization (CI) GC Quadrupole Quadrupole Ion Trap Linear Quadrupole Ion Trap Magnetic Sector Time of Flight (ToF) Orbital Ion Trap (Orbitrap) Fourier-Transform Ion Cyclotron Resonance (FT-ICR) Quadrupole Quadrupole Ion Trap Linear Quadrupole Ion Trap Magnetic Sector Time of Flight (ToF) Orbital Ion Trap (Orbitrap) Fourier-Transform Ion Cyclotron Resonance (FT-ICR) Electrospray Ionization (ESI) Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure Photoionization (APPI) LC Highlight Q Tof

Electron Ionization (EI) Mass Spectrometry Electron Ionization (EI) Beam of electrons at about 70eV strip the analyte (M) of an electron This leaves the ionized analyte (+) with an odd number of electrons If this radical has enough energy, it can fragment in a characteristic pattern In some cases the intensity of the molecular ion can be low.

Ionization in GC: chemical vs electron Mass Spectrometry Ionization in GC: chemical vs electron Chemical Ionization (+) Electron Ionization (+) [M+H]+ [M+28]+ [M+40]+ Accurate mass [u] 397.1690 Mass accuracy [ppm] 5 Isotopic abundance error [%] 5 A+1 [%] 37.90 A+2 [%] 17.84 A+3 [%] 5.03 Electron Ionization: Very typical fragmentation pattern at 70eV Chemical Ionization: [M+H]+ is very abundant (“soft ionization”) Example: adduct ions at M+28.02=[M+C2H5]+ and M+40.04=[M+C3H5]+ are used for verification of [M+H]+ Different ionization gases can be used such as NH3, methane, butane  different adducts

Electrospray Ionization (ESI) Mass Spectrometry Electrospray Ionization (ESI) Arrows must follow

Mass Spectrometry Ionization Shorten to esi and ei

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

Mass Spectrometry Spectrum relative abundance m/z Base peak Fragment Molecular ion m/z

Total Ion Current (TIC) vs Spectrum Detection Total Ion Current (TIC) vs Spectrum Normalized Intensity 100 50 75 25 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 30 40 60 70 80 90 110 120 130 140 150 160 170 Peak selector Chromatogram (GC-MS) Time [min] Normalized Intensity 166 Mass spectrum (EI) 97 129 61 83 47 35 70 112 119 m/z

Mass Spectrometry Matrix Effects Unwanted effects that modify (often decrease) the MS signal Exact mechanisms unknown, but likely include: The competition of co-eluting molecules for charge during ionization In LC – changes in the composition of the droplet surface during evaporation Every matrix is different Absolute quantification can be achieved by standard addition Standard curve in your sample Matrix effects can be minimized by proper sample preparation

Mass Spectrometry Mass Spectrometers • There are several types of mass spectrometers: - TOF (time of flight) - Q, QQQ (quadrupole) - Ion Trap - Orbitrap - FTICR (Fourier transform ion cyclotron resonance) The goal is to separate ions based on mass-to-charge ratio Quad TOF

Definitions and concepts Mass Spectrometry Definitions and concepts • Isomer- compounds with the same chemical formula e.g. propanol and isopropanol (C3H8O) C8H10N2O has 100,082,479 isomers Isotopes vs isobar What are isobars • Isotopes- nuclei with different numbers of neutrons in their nuclei e.g. 12C vs 13C • Isobaric compounds- compounds with similar masses e.g. CO (27.9949) and C2H4 (28.0313)

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

Why is resolution important? 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 average mass -accurate mass can distinguish isobars, not isomers

Mass Spectrometry ToF

Mass Spectrometry MS/MS

Mass Spectrometry MS/MS Product Ion Scan Select specific m/z in Q1 Collisions in q2 Scan in Q3 Useful for helping to definite structure/class of compounds Precursor Ion Scan Scan m/z in Q1 Collisions in q2 Select specific m/z in Q3 Useful for helping to measure compounds of a specific class Fix mass

Mass Spectrometry MS/MS

Mass Spectrometry MS/MS Neutral Loss Scan Scan a specific range in Q1 from x1….xn Collisions in q2 Scan in Q3 equal to (x1-a)….(xn-a) Useful if m/z = a is an interesting conjugate/derivative. For example, if you are looking for glycosides. Single/Multiple Reaction Monitoring (SRM/MRM) Select specific ions in Q1 and Q3 with collisions in q2 (SRM) Useful for specifically measuring known compounds. Reduces noise from compounds with the same mass that may fragment differently. Several reactions can be monitored at the same time (MRM) Fix mass

Mass Spectrometry Neutral Loss Lightning bolts beside circles

Instrument Parameters Mass Spectrometry Instrument Parameters Mass range The m/z over which the instrument is useful Determines whether an analyte can be measured Mass accuracy Deviation from calculated m/z Mass resolution Ability to tell isobars apart Linear dynamic range The concentration range over which a linear response is obtained. Determines the capability of an instrument to do quantitative analysis Speed (acquisition rate) The number of spectra that can be acquired per second 1 scan/ sec = very slow 500 scans/sec = very fast Sensitivity Lowest amount an instrument can detect

Why is high speed important? Mass Spectrometry Why is high speed important? In order to deconvolute (separate/clean) overlapping peaks, enough mass spectra have to be acquired to perform the mathematical calculations. With only one spectrum per second this is impossible. That requires: a) fast scanning detectors like time-of-flight (TOF) b) fast data acquisition hardware/software (DAC/ADC) The LECO TOF can acquire up to 500 mass spectra per second. For GC-MS 20 spectra/second sufficient For comprehensive GC (GCxGC) up to 200 spectra/sec needed Source: LECO ChromaTOF Helpfile

Properties of various mass spectrometers Mass Spectrometry Properties of various mass spectrometers   TOF Quad Ion Trap Orbitrap FT-ICR Resolving Power very good fair excellent Dynamic Range Sensitivity Speed good Cost 150-300K 100K 500K 1M Maintenance ave very high

Mass Spectrometry There is no perfect mass spectrometer Sensitivity and resolution are m/z dependent Differences can vary based on the type of mass spectrometer used Different applications call for different specifications. Metabolome contains very different compounds, which cannot all be accurately measured concurrently not only because of: Sampling Extraction Chromatography But also mass spectrometry itself. Q-ToF Orbitrap Resolution (FWHM) m/z

Homework Use what you have learned about mass spectroscopy to answer the following questions: You find a parent ion with m/z = 760.4861. How do you determine its identity? How would you search for an expected compound in a MS dataset (e.g. sucrose)?