1. Metabolomics Part 2 Mass Spectrometry
PCB 5530
Fall 2017

2. Metabolomics Class I Important Points
What are the major reasons metabolomics is much more difficult than genomics or proteomics?
Number of molecules (>105)
Chemical diversity
What is the most important part of a metabolomics experiment?
Sample preparation
What are the two types of chromatography used in metabolomics?
GC & LC
What is the largest limitation of each?
GC – limited to compounds that are (or can be modified to be) volatile and thermostable
LC – limited availability of standard protocols and databases

3. Mass Spectrometry in Metabolomics
Definitions and important concepts
Principle Components of a Mass Spectrometer
Overview
Ionization
Common complications and problems
How a time of flight instrument works
Important mass spectrometer parameters

4. Definitions Ion – a charged molecule (either + or −)
m – the mass of an ion
z – the charge of an ion (the number of + or −)
m/z – the mass to charge ratio
What a MS measures
Average mass vs the mass of an atom
Average takes into account of the isotopic distribution on earth of a particular atom
Carbon is , but 12C is defined as exactly 12.
Glucose C6H12O6 – g/mol exact mass g/mol
Average mass – sigma says glucose is

5. Mass Spectrometry The Principles
Weighs charged molecules
Ions can be manipulated at a distance by electric and magnetic fields
Ions must be in gas phase
Whole instrument must be in a vacuum
Otherwise the ions will bump into other molecules
Orbitrap <10-9 mbar
Atmosphere 1013 mbar
The 1935 Labor Day hurricane, with a pressure of 892 mbar – Irma 915 mbar

6. Mass Spectrometry The Principles
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)

7. Mass Spectrometry Spectrum Base peak – most abundant Molecular ion
Fragment
C6H6 = 78
[M+H]+

8. 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 ( ) and C2H4 ( )

9. 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 m/z)

10. 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

11. A Mass Spectrometer 1. Sample 2. Ion Source 3. Mass Analyzer
4. Detector
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 3 2 1 4

12. 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.

13. Ionization in GC: chemical vs electron
Mass Spectrometry
Ionization in GC: chemical vs electron
Chemical Ionization (+)
Electron
Ionization (+)
[M+H]+
[M+28]+
[M+40]+
Electron Ionization: Very typical fragmentation pattern
[M+H]+ is minor
Chemical Ionization: [M+H]+ is very abundant (“soft ionization”)
Because it involves a chemical reaction, the adducts that form can help one identify the molecular ion (or [M+H]+ )

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

15. Mass Spectrometry Ionization Proteomics Metabolomics
Shorten to esi and ei

16. Mass Spectrometry Spectrum Base peak – most abundant Molecular ion
Fragment
C6H6 = 78
[M+H]+

17. 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]+)

18. 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

19. Major Complications to MS-based Metabolomics
Mass Spectrometry
Major Complications to MS-based Metabolomics
Unwanted effects that modify (often decrease) the MS signal
Matrix Effects
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
Matrix effects can be minimized by proper sample preparation

20. Major Problems in MS-based Metabolomics
Mass Spectrometry
Major Problems in MS-based Metabolomics
Sample preparation
Always a trade-off between metabolite recovery, comprehensiveness and time.
Inadequate sample preparation can result in large biases and problems with reproducibility.

21. Mass Spectrometry There are many different types of mass spectrometers
Magnetic sector
ToF (Time of Flight)
Ion trap
FTICR (Fourier transform ion cyclotron resonance)
Q, QQQ (Quadrupole)
Orbitrap
But they all do the same thing – separate ions based on mass and measure their abundance
There are several types of mass spectrometers:
- TOF (time of flight) - Q, QQQ (quadrupole)
- Ion Trap - Orbitrap
- FTICR (Fourier transform ion cyclotron resonance)

22. Time of Flight (ToF) Mass Analyzer
Mass Spectrometry
Time of Flight (ToF) Mass Analyzer
Based on Newton’s second law of motion F=ma
If a constant force is applied, the acceleration will be proportional to the mass
Therefore, if you have a set of ions travelling in a constant magnetic field (that applies a constant force):
a ∝ m/z
and
v ∝ m/z
Thus, the time it takes an ion to reach the detector will also be proportional to its m/z

23. Mass Spectrometry
ToF

24. Resolution is important!
Mass Spectrometry
Resolution is important!

25. 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

26. 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

27. Mass Spectrometry MS with accurate mass: ToF Orbitrap FT-ICR
Advantages
Determine the elemental composition of an analyte
And its fragments
Distinguish between isobars
Disadvantages
Scan time
Sensitivity
Cost
Make a list of accurate mass and not accurate mass

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