1. Metabolomics Part 2 Mass Spectrometry
PCB 5530
Guillaume Beaudoin
Fall 2016

2. 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)

3. 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

4. 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.

5. 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]
Mass accuracy [ppm] 5
Isotopic abundance error [%] 5
A+1 [%]
A+2 [%]
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

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

7. Mass Spectrometry
Ionization
Shorten to esi and ei

8. 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]+)

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

10. 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

11. 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

12. 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

13. 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 ( )

14. 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)

15. 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

16. Mass Spectrometry
ToF

17. Mass Spectrometry
MS/MS

18. 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

19. Mass Spectrometry
MS/MS

20. 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

21. Mass Spectrometry
Neutral Loss
Lightning bolts beside circles

22. 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

23. 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

24. 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 K
100K
500K 1M
Maintenance
ave
very high

25. 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

26. 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)?