Mass spectrometry

and related
technologies.
James Dalton Ian Donaldson
Rhidhwan Wahab Qian Zhen

MRes Bioinformatics. 2002.

University of Leeds, UK.

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 Mass Spectrometry
By James Dalton.
What is Mass Spectrometry?
What information can it provide?
Where are Mass Spectrometers used?
The biochemical applications
How does a Mass Spectrometer work?
Some nice internet sites

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 What is Mass Spec?
Analytical tool measuring molecular weight
(MW) of sample
Only picomolar concentrations required
Within an accuracy of 0.01% of total weight of
sample and within 5 ppm for small organic
molecules
For a Mr of 40 kDa, there is a 4 Da error
This means it can detect amino acid
substitutions / post-translational modifications

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What sort of info is returned?

Structural information can be generated

Particularly using tandem mass
spectrometers
Fragment sample & analyse products
Useful for peptide & oligonucleotide
sequencing
Plus identification of individual compounds in
complex mixtures

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 Where are they used?
Biotechnology:
analysis of proteins, peptides, oligonucleotides
Pharmaceutical:
drugs discovery, combinatorial chemistry, pharmokinetics,
drug metabolism
Clinical:
neonatal screening, haemoglobin analysis, drug testing
Environmental:
water, food, air quality (PCBs etc)
Geological:
oil composition

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 In biochemistry
Accurate molecular weight measurements:
purity of sample, detection of amino acid substitutions, post-
translational modifications, and disulphide bridges
Reaction monitoring:
enzyme activity, chemical modification, protein digestion
Amino acid sequencing
Oligonucleotide sequencing
Protein structure:
protein folding (H/D exchange), protein-ligand complex
formation, macromolecular structure determination

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 How does a Mass
Spectrometer work?
3 fundamental parts: the ionisation source,
the analyser, the detector
Samples easier to manipulate if ionised
Separation in analyser according to mass-to-
charge ratios (m/z)
Detection of separated ions and their relative
abundance
Signals sent to data system and formatted in
a m/z spectrum

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 Simplified Schematic

The analyser, detector and ionisation source are

under high vacuum to allow unhindered movement of
ions
Operation is under complete data system control

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m/z spectrum example

Leucine Enkephalin, Pentapeptide, YGGFL

Expected Mr = 555.3 Da, Calculated Mr = 555.1 Da (dominant
ions at 556.1)

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 Sample Introduction
& Ionisation
Direct into ionisation source or via
chromatography for component separation
(HPLC, GC, capillary electrophoresis)
Ionisation can be positively charged (for
proteins) or negatively charged (for
saccharides and oligonucleotides)

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 Ionisation methods
Atmospheric Pressure Chemical Ionisation (APCI)
Chemical Ionisation (CI)
Electron Impact (EI)
Electrospray Ionisation (ESI)
Fast Atom Bombardment (FAB)
Field Desorption / Field Ionisation (FD/FI)
Matrix Assisted Laser Desorption Ionisation
(MALDI)
Thermospray Ionisation

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Detection & Recording of Ions

Detector monitors ion current, amplifies

it and then transmits signal to data
system
Common detectors: photomultiplier,
electron multiplier, micro-channel plate

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 Peptide Sequencing
Peptides of 2.5 kDa or less give best data
Protein sample often taken from 2-D gels and digested
A protein digest can be analysed as entire mix
Initial MS spectrum showing Mr of all components in digest
(peptide map) may be enough for a database search and
identification
Peptides fragmented along the amino acid backbone in tandem
mass spectrometry
Some peptides generate enough info for full sequence, others
only generate partial sequences of 4-5 amino acids
Often this “tag” sequence is sufficient for database identification

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 Internet sites
www.astbury.leeds.ac.uk/Facil/MStut/mstutorial.htm
(Dr Alison E. Ashcroft at Leeds)
www.asms.org (The American Society for Mass Spectroscopy)
www.spectroscopynow.com (Base Peak)
Mass Spec tools
www.expasy.ch/tools/#proteome
http://prowl.rockefeller.edu
www.mann.embl-heidelberg.de

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Introduction to Tandem Mass
Spectrometry. By Ridhwan Wahab.

Mass spectrometry is a very powerful method to

analyse the structure of organic compounds, but
suffers from 3 major limitations:

1 Compounds cannot be characterised without clean

samples

2 This technique has not the ability to rovide sensitive

and selective analysis of complex mixture

3 For big molecules like peptides spectra are very

complex and very difficult to interpret
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MS/MS allowed to solve these problems.

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 History of the technique

MS/MS or tandem mass spectrometry or 3D MS was first

introduced in the 1970’s and was quickly accepted in
analytical community.

The technique has been used for structure elucidation of

unknown and for analysis of complex mixture with
minimum sample clean up.

Development in the mid-1980’s and advancing the

popularity of MS/MS included the availability of powerful
data system able to control the MS/MS experiment.

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 Principle

The introduction of the complex mixture in the inlet of a

mass spectrometer always gives a complex spectrum
which is difficult to interpret.

We often observe a lot of peaks and the most important

correspond to the major constituent. That’s why it is very
difficult to identify one precise compound in the spectrum of
a mixture.

The principle of MS/MS method is the

use of Filiation Reactions. This method
introduce a further specificity to
recognise a substance in a mixture.

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In Tandem MS – normally has 2 mass spectrometers in series.

In first mass spectrometer (MS1) is used to SELECT, from the

primary ions, those of a particular m/z value which then pass
into
the Fragmentation Region. The ion selected by the MS1 is the
parent ion and can be a molecular ion resulting from the primary
fragmentation. DISSOCIATION occurs in the fragmentation
region. The daughter ions are analysed in the Second
Spectrometer (MS2). In fact, the MS1 can be viewed as an ion
source for MS2.

MS1 MS2 19
• In MS1, ionisation is made by electron impact. Then one ion is selected by
a particular system (quadrupole, magnetic sector, ion cyclotron resonance).
This only one type of ion go through the outlet to the fragmentation region.

• In the fragmentation region, there is a neutral gas (i.e He, Ar, N2….) on a
high pressure. The ions selected interact with these molecules of gas. During
the collision, the kinetic energy of ions is transformed into internal energy
which permits the fragmentation into daughter ions.

• In MS2, the daughter ions are detected and the final spectrum shows the
peaks of selected ion and all its daughters.

The MOST
important peaks

appear in the front and daughter peaks

behind them. 20
Instrumentation for MS/MS

A tandem mass spectrometer can be thought of as two mass spectrometers

in series connected by a chamber that can break a molecule into pieces
perhaps like a puzzle. This chamber is known a collision cell.

Collision
Source MS1 MS2 Detector
Cell

Every m+1 Intact m+1 plus Ion separation

m+ fragment ions

A sample is “sorted” and “weighted” in the first mass

spectrometer (MS1), then broken into pieces in the collision cell,
and a piece or pieces sorted and weighted in the second mass
spectrometer (MS2).

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Types of sources

• ESI (Electrospray)
• FAB (Fast Atom Bombardment)
• LSI (Liquid Secondary Ion)
These terms indicate the way the compound is placed into the tandem mass
spectrometer.

Dissolvation-nebulization sources : ESI (“electrospray”)

(flow rate : 1 to 10 mL/min).

Different parameters of “spray” also exist :

“ Thermospray ” source
“ APCI ” (Atmospheric Pressure Chemical Ionisation)
“ Particle beam ” source
“ Nanospray ” (2 to 20 nL/min)
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Types of sources

Ionisation-desorption sources
Based on the secondary emission :
the bombardment of a solid or liquid sample with a primary beam (ion, atom or
photon) induce the secondary emission of particles : electrons, neutral particles
and ions. Only these particles are analysed by mass spectroscopy.

The name of the sources depends on the nature of the incident beam :
SIMS (“Secondary Ion Mass Spectrometry”), ION
FAB (“Fast Atom Bombardment”), ATOM
LD (“Laser Desorption”), PHOTON
MALDI (“Matrix Assisted Laser Desorption Ionisation”)

In FAB, the sample can be mixed with the matrix.

(This type of sources are used to analyse compounds which have a high
molecular weigh, especially the polymers and the biological compounds)
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Analysers

A tandem mass spectrometer is a mass spectrometer that has more than

one analyser, in practice usually two. The two analysers are separated by a
collision cell into which an inert gas (for example, argon or xenon) is
admitted to collide with the selected sample ions and brings about their
fragmentation.

Most Common combinations being:

• quadrupole-quadrupole
• magnetic sector – qudrupole
• magnetic sector – magnetic sector
• quadrupole- time-of-flight (TOF)

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Tandem Quadrupoles

TQMS are the most commonly used : it’s a sequential arrangement of two
mass analysing quadrupoles with a third quadrupole interposed as a gas
cell.

A quadrupole is made up of four electrodes where a continued potential U

and a radio-frequency potential Vcos (wt) are applied.
(Each ion adopts an oscillating path whose amplitude depends on the ratio U/V
and on the ratio m/z. We can stabilize or destabilize the trajectory of ions with
scanning of U and V but with the ratio U/V which is always constant. Only ions
which have a stable trajectory can be detected.)

The first quadrupole (MS1) allows the selection of precursor ions according
to m/z value. The third sector MS2 is a mass filter too for the daughter ions.
In the intermediate quadrupole, there exists a high pressure.
This role is to induce ionic oscillations for ions to have an important linear speed.
This quadrupole is not selective according to the mass, it is a RF-only quadrupole
(alternative alimentation of radio-frequency).
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Sector Mass Spectrometers

Ion kinetics energies are fundamental importance to the mass analysing

properties of sector mass spectrometers.
They are double focusing instruments and they use a magnet (B) with electric
sector (E). When the electric sector takes place between the source and the
magnet, the geometry is normal (EB) and when it is between the magnet and
the detector, the geometry is inverse (BE), for example MIKES (Mass analysed
Ion Kinetic Energy Spectrometry).
In the EB system : The ions issued from source are placed into electric sector
where they are accelerated by a potential V. Then, they are placed into a
magnetic sector where the magnetic field is perpendicular to the direction of
their movement. So, the ions describe an arc of the circle.
In practical, scanning the magnetic sectors allows to focus the ion
fragments on the slit of the analyser. The analysers with electric and
magnetic sectors have a good resolution but are too big and expensive.

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Ion Trap
The principle of Ion Trap is next to this of tandem quadrupoles. The
difference is that ions can be formed directly in the Trap : so, the
source and the analyser are the same instrument. The excellent
sensibility of the Ion Trap involves its use to detect and quantify traces.
This technique is relatively new and we can find it in environmental
laboratories (analysis of pesticides, micro pollutants).

Ion Cyclotron Resonance (ICR)

It is the same principle as Ion Trap. With this type of analyser, we can
study ions which have a ratio m/z bigger than 5000. The pressure
inside the cell is low (10-9 torr). The performances of ICR are
promising but they are very expensive.

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Collision cell

The collisional activation can be divided into two categories,

involving high or low energy, to which different types of collision
cell are appropriate.

In sector instruments, where high energy collisions are most

common, the cell is usually a tight chamber of 1-3 cm length with
entrance and exit slits which transmit the ion beam. Good
pumping is essential to maintain a low pressure outside the cell.

In some instruments, the collision cell has been electrically insulated

from the mass spectrometer and can be held at a high potential to
retard the ion beam and reaccelerated it on exit. This allows control of
the collision energy and also reduces the kinetics energy spread of
daughter ions formed.

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Types of detector
The detector’s purpose is to translate the ion arrival into an electric signal
measured by the electronic system of the mass spectrometer. It exists
two different classical types of detector.

1. Electrons multiplying
The principal sorts of electron multiplying are the “channeltron” and the
“micro channel plate”. The principle is based on the impact of an ion
on a surface composed by half conductors. This impact creates
electrons which are accelerated to another surface where they also
create other electrons, etc…Those electrons are recovered and the
number of them is proportional to the signal’s intensity.

2. Photodetectors
Electrons are created in the same way and interact with a
phosphorecent surface which generate photons. Those photons are
also recovered and the number of them is proportional to the
signal’intensity.
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Applications of Tandem Mass Spectrometry

Analysis of Food constituents

• Labows and Shuhan (1983) - volatiles substances (i.e limonene/ethyl
butyrate).
• Warburton (1981) - identification of cholesterol in egg york & citric acid in
lemon.
• Walther (1983) - presence of caffeine in leaf tea.

Protein identification

Structural studies
• Sherman et al. (1985)- phosphoinositides, representing an important
class of glycerolphospholipids.

Analysis of drug residue

• International Olypic Committee - detection of anabolic steroids such as
stanozol [10418-03-8].

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Applications of Tandem Mass Spectrometry

Analysis for contaminants

• Plattner and Bennett (1983) - rapid detection of mycotoxins in food and
animal feeds.
• Steiner et al (1980) - detection of parathion (pesticides residues in food)
• Lau et al (1985) - identification of arsenobetaine and arsenocholine
(organometallic compounds) in fish.

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Tandem GC/MS/MS

SRM: (Single Reaction Monitoring)

MRM:(Multiple Reaction Monitoring)

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Bibliography

Internet sites :

 http://www.google.com
• http://www.bmss.org.uk/what_is/whatis.html
• http://www.duke.edu/~mdfeezor/NSHome/inform/msms1.html
• http://www.astbury.leeds.ac.uk/Facil/MStut/mstutorial.htm
• http://ms.mc.vanderbilt.edu/tutorials/ms/3.htm
• http://www.garvan.unsw.edu.au/public/corthals/book/IPMS.html
• http://www.micromass.co.uk/basics/Glossary.html

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Ionization Methods
—MALDI and ESI
QIAN ZHEN

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 Ionization Methods
Sort of ionization methods
APCI—Atmospheric Pressure Chemical Ionization
CI—Chemical Ionization
EI——Electron Ionization
ESI—Electrospray Ionization
FAB—Fast Atom Bombardment
FD—Field Desorption
FI—Field Ionization
MALDI—Matrix Assisted Laser Desorption
Ionization
TSP—Thermospray Ionization

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Ionization Methods

Two Samples
—MALDI and ESI
Main Advantage: Soft Ionization

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 Ionization Methods

Soft Ionization: They do not (in the most

part) degrade the molecule during the
ionization process.

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 Ionization Methods
—MALDI
http://www.fbsmres.leeds.ac.uk/users/bmbqz/5070/Movie4.swf

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 Ionization Methods
—MALDI

The Mechanism of MALDI

—Ion Desorption
The Formation of a ‘Solid Solution’
Matrix Excitation
Analyte Ionization

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 Ionization Methods
—MALDI
Advantage:
Rapid and convenient molecular weight determination.

Limitations:
1) MS/MS difficult.
2) Requires a mass analyzer that is compatible with
pulsed ionization techniques.
3) Not easily compatible with LC/MS.

Mass range: typically less than 500,000 Da

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 Ionization Methods
—ESI
http://www.fbsmres.leeds.ac.uk/users/bmbqz/5070/Movie5.swf

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 Ionization Methods
—ESI

The Mechanism of ESI

—Ion Evaporation

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 Ionization Methods
—ESI
Advantages:
1) Good for charged, polar or basic compounds.
2) Permits the detection of high-mass compounds at mass-to-
charge ratios that are easily determined by most mass
spectrometers (m/z typically less than 2000 to 3000).
3) Best methods for analysing multiply charged compounds.
4) Very low chemical background leads to excellent detection
limits.
5) Can control presence or absence of fragmentation by
controlling the interface lens potentials.
6) Compatible with MS/MS methods.

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 Ionization Methods
—ESI
Limits:
1) Multiply charged species require interpretation and
mathematical transformation.
2) Very sensitive to contaminants such as alkali metals or
basic compounds.
3) Relatively low ion currents.
4) Relatively complex hardware compared to other ion
sources.

Typically less than 200,000 Da.

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 Ionization Methods
—Applications
1) Proteins and peptides:
- Monitoring biomolecular reactions.
- Analysis of protein conjugates.
- Analysis of high molecular weight biopolymers.
- Nested-PSD for interpretation of peptide sequences.
2) Polymers.
- Analysis of synthetic polymers.
- Analysis of epoxy resins.
- Structural determination in MALDI by examination of the
Neutral Spectrum.

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 Ionization Methods
Further Reading
1. For MALDI beginner:
http://www.srsmaldi.com/Maldi/Guide.html
2. For MALDI lab user:
http://www.srsmaldi.com/Maldi/Lab.html

3. For MALDI tutorial:

http://ms.mc.vanderbilt.edu/tutorials/maldi/maldi-ie_files/frame.htm

4. Ionization Methods 1:
http://www.jeol.com/ms/docs/ionize.html
5. Ionization Methods 2:
http://www.waters.com/Waters_Website/Applications/lcms/lcms_itq.htm

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 By Ian Donaldson.

Surface-enhanced
laser desorption/ionization-
time of flight-mass spectrometry.
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What is SELDI-TOF-MS?
• Surface-Enhanced Laser Desorption/Ionization-
time of flight-mass spectrometry.
• Advantages over the MALDI technology:
- Use of complex biological material
- No pre-processing or labelling
- Chromatography on-chip
- Uses small amounts (< 1l/ 500-1000 cells) of
sample (biopsies, microdissected tissue)

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ProteinChipTM by Ciphergen Biosystems Inc.
• Sample fractionation accomplished by ‘Retentate’
chromatography.
• Sample deposited on spots, each chip has different
surface interactions.

Chromatographic: Biological:
- Cation exchange - Antibody/Antigen

- Anion exchange - Receptor/Ligand

- Metal affinity capture - DNA/Protein

- Normal phase - Enzyme

(Hydrophobic) (bound to preactivated surface)
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ProteinChipTM.
Protein purification by ‘Retentate mapping
chromatography’.

Slide adapted from http://www.ciphergen.com/tech_doc11.2.html

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SELDI process.

1. Add crude extract

2. Washing

3. Add EAM

4. Detection by TOF-MS

EAM = Energy absorbing molecule

gifs from http://www.bmskorea.co.kr/new01_21-1.htm
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Applications of SELDI?
• Fingerprinting/Biomarker discovery
Detect proteins from two different states e.g. disease versus
normal tissue samples.
• Toxicology
Detection of toxicity biomarkers
• Protein characterisation
Including the identification of novel ligands and protein
characteristics.
• Assay development
Simplification or advancement of existing methodologies.
On-chip peptide mapping with trypsin digestion (ProFound).
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 SELDI ProteinChipTM Arrays:
-----for Epitope Mapping
Step 4: Bound epitopes
Step 3: Fragments (outside of Ab
identified by
binding site) removed
SELDI

Step 1: Bind protein antigen(s) Wash away unbound fragments

to affinity capture device (Ab)
on ProteinChip™ array Bound epitopes retained

Step 2

In Situ
Digest

Definition of Antigenic Determinants: Epitope Mapping

Slide adapted from http://www.ciphergen.com/images/slide.PPT
53
ProteinChipTM Software Analysis.
• Data Acquisition.
- Protocol design
- Automated mass data collection and creation of a
‘retentate map’.
• Data views.
Several presentation formats (‘data views’).
- Spectrum view or ‘retentate map’
- Peak map
- Difference map view
- Gel view
- 3-D overlays
- Internal protein calibrants (based on pI/MW)

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Difference map view.

Gel view.

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SELDI: Summary.
Improvements over existing technologies:
• Small amount of starting material
• Sample does not need to be processed
• Versatile applications and analysis

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Selected reviews on SELDI:
• Merchant M and Weinberger S (2000) Recent advancements in
surface-enhanced laser desorption/ionization-time of flight-
mass spectrometry. Electrophoresis 21: 1164-1167.
• Fung E, Thulasiraman V, Weinberger SR, Dalmasso EA (2001)
Protein biochips for differential profiling. Current Opinion in
Biotechnology 12: 65-69.
• Weinberger SR, Dalmasso EA, Fung ET (2002) Current
achievements using ProteinChipTM array technology. Current
Opinion in Chemical Biology 6: 86-91.

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SELDI Web sites:
• Molecular Analytical Systems (MAS).
http://www.seldi.org/
• Manufacturers of ProteinChip(R)
http://www.ciphergen.com/

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