Principles and Applications

of Mass Spectrometry

BS221 2008
University of Essex

 Introduction to mass spectrometry

.

Basics concepts and terminology

Instrumentation, ionization modes
and types of mass analyzers
Applications of mass spectrometry
to the study of biological molecules

 Mass spectrometry studies ions

Mass spectrometers determine
mass-to-charge ratio of ions
The analysis is performed in the
gas phase not in solution
Structure can be elucidated by
determining the mass of fragments

 Mass spectrometry generates mass spectra

A mass spectrum is a set of peaks one for
each ion that has been detected
Peaks are characterized by their m/z
The height of the peak depends on the number
of ions of that particular m/z that has hit the
detector. This number is referred to as intensity

Monoisotopic and average mass

Because of the presence of natural isotopes in

the biological molecules we distinguish two types
of mass measurements monoisotopic and
average

Resolution and accuracy

Resolution is defined as the ratio of the peak

mass divided by its width at half maximum. It can
also be expressed in FWHM units.
FWHM = Full Width at Half Maximum.
Mass accuracy is the measure of the error
relative to the theoretical value

General design of a mass spectrometer

and MALDI

Ion Source

Mass Analyzer

Detector

Data Station

Types of ionization
Electron Impact - sample is heated and energized by
a beam of electrons, usually gives a molecular ion
(M+) and a lot of fragments

Chemical Ionization sample is heated and ionized

in the presence of Cl and proton donors. Gives (M+H)
+ ions and less fragments compared to EI

Fast Atom Bombardment softer than EI and CI.

Ions are produced by bombardment with heavy
atoms. Gives (M+H)+ ions and litle fragmentation.
Good for more polar compounds.

Types of ionization
Matrix Assisted Laser Desorption Ionization
(MALDI) sample is co-crystallized with a matrix and
then irradiated with laser. Singly charged (M+H)+ and
(M-H)- ions are produced. Good for intact proteins.

Types of ionization
ElectroSpray Ionization sample is sprayed out of a
narrow nozzle in a high potential field. Generates
positive (M+nH)n+ and negative (M - nH)n- ions and
almost no fragmentation. Generates multiple charged
ions.

Types of mass analyzers

Magnetic sector analyzer Uses electric and/or
magnetic fields to separate ions
Time of Flight analyzer (TOF) ions are accelerated
through a flight tube and the time of light to the
detector is measured
Quadrupole analyzers ions are filtered or trapped in
a device consisting of several metal rods using
specifically tailored electromagnetic fields
Fourier Transform Ion Cyclotron Resonance (FT ICR)
analyzers very expensive. Use a very high strength
magnet to trap ions in a circular orbits and measure
their resonance energies. Provide the highest
resolution and sensitivity

Magnetic sector instruments

Ions are deflected and accelerated down a curved path to

the detector.

Time of flight instruments

E = (mv2)/2

(1)

v = (2E/m)1/2

(2)

Ions are accelerated and their time of flight to the detector

is measured

Quadrupole analyzers

Ions are filtered by application of RF fields on the

quadrupole. The analyzer scans sequentionally through
the mass range by varying the RF potential

Quadrupole ion trap analyzers

Ions are trapped by an
oscillating RF voltage
and scanned out
sequentially by changing
the RF voltage. Collision
with He helps to cool the
ions and focus them in
the centre of the trap.
Ion traps are cappable
of MSn operation.

More about Ion Traps

More about Ion Traps

Ion trap geometry

Mathieu stability diagram

Tandem and Hybrid analyzers

TOF-TOF

Q-TOF

Two consecutive tof analyzers

with a collision cell in between

A quadrupole mass filter

followed by a collision cell and
a tof analyzer

Two consecutive quadrupole

analyzers with a collision cell
in between

Triple Quad

MS/MS and MSn

Full scan

Precursor isolation

Fragmentation

Detection

Auto MS/MS
The instrument alternates
between MS and MS/MS
mode selecting precursors
according preset criteria

Selected precursors are

added to the include list
to force their isolation and
analysis

SIM and SRM

The instrument isolates just one predetermined ion,
fragments the isolated precursor (in SRM) and then
measures the intensity of a selected fragment ion. This
is very powerful quantification method. It is extremely
specific and sensitive
Several peaks with the same m/z
which one is the real compounds

Both usually require separation by

HPLC prior to MS. SRM has much
higher specificity
MRM simultaneous SRM of several
precursors

MS analysis of biomolecules
Small molecules: drugs, drug breakdown products, sugars, lipids, amino
acids, fatty acids and their esters
EI and CI (chemical ionization), ESI, are used predominantly in combination
with sector, quadrupole and time-of-flight mass analyzer

Biopolymers: proteins, peptides, oligonucleotides, carbohydrates

ESI and MALDI ionization mostly. All mass analyzers