M.

Prasad Naidu
MSc Medical Biochemistry, Ph.D,.
Mass spectrometry (MS) is an analytical technique
that measures the mass-to-charge ratio of charged
particles.

It is used for determining masses of particles, for
determining the elemental composition of a sample
or molecule, and for elucidating the chemical
structures of molecules, such as peptides and
other chemical compounds
The sample is ionised to generate parent
molecular ions

Further fragmentation will generate fragment ions

The process of ionisation has to be controlled to
generate similar ions from all the molecules in the
mixture





The ions are separated according to their mass-to-
charge ratio in an analyzer by electromagnetic fields

The ions are detected, usually by a quantitative
method

The ion signal is processed into mass spectra








The analyser, detector and ionisation source are under
high vacuum to allow unhindered movement of ions
Operation is under complete data system control

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


Prior to sample introduction, 2 things must be
achieved:
Sample must be introduced into vacuum
Sample must be vaporized

The sample is introduced by placing it on the probe,
which is then inserted through a vacuum lock into
ionisation region of mass spectrometer
Ionization means placing a charge on a
neutral molecule

Methods:

1) Electron ionization
2) Electrospray
3) Matrix-assisted laser desorption/
ionization(MALDI)

Also known as electron bombardment / electron
impact method

The sample is heated to vaporize it

The sample in the gas phase is now delivered into
electron ionization region

Here a beam of electrons with energy of 70EV is made
to interact with the sample

This interaction causes electron ejection in the sample
molecules leading to ionization

Generates ions directly from aqueous or
aqueous/organic solutions

The solution is forced through a narrow needle which is
kept at a high potential (3.5 kV)

The voltage on the needle causes the spray to be
charged as it is nebulized

Thus, very small droplets are created and they are
charged on their surfaces

The electric charge density on the surface of the droplet
is a function of its size- smaller the droplet, larger is the
electric charge density

Thus, as the droplets decrease in size, there is repulsion
between mutually charged droplets

At this point, ions begin to leave the droplet

Ions are led into mass analyzer

It nondestructively vaporizes & ionizes both big and
small molecules

The analyte is first co-crystallized with an excess of a
matrix compound

Matrix compounds are organic acids, which absorb in
the UV range

After the co-crystallization, a pulse UV laser beam is
focused on the surface of the crystal

The matrix absorbs the radiation & is vaporized

The analyte is also vaporized and carried along with the
matrix

The matrix doubles up as a proton acceptor or donor &
thus also ionizes the analyte

Different matrices:

2,5 dihydroxy benzoic acid-proteins,peptides &
oligonucleotides

Sinapinic acid proteins & peptides


After ionization, ions that are in the gas phase enter the
mass analyzer

It separates ions within a selected range of mass-to-
charge (m/z) ratios

To separate ions,different mass analyzers use magnetic
fields,electric fields or the time taken by an ion to move
over a fixed distance
J.J Thompson,who built the 1
st
mass spectrometer, used a
magnet to measure the m/z value of electrons

It separates ions in a magnetic field according to the
momentum & charge of ion

A 1 to 10kV electric field accelerates ions from the source
region into the magnetic sector

Once it reaches the magnetic field,the ion beam is bent in
an arc by the magnetic field
The radius of the arc(r) depends on:
Momentum of the ion
Charge of the ion
Magnetic field strength

The greater the momentum of the ions, the larger is
their arc radius

The separation of ions is thus based upon their
momentum

Hence, magnetic analyzers are also called momentum
analyzers
In the mass spectrometer, an electric field accelerates
ions out of the source region and into the quadrupole
analyzer

The quadrapole analyzer consists of 4 rods/electrodes
arranged across from each other

The ions are made to travel through the quadrupole

Here, they get filtered according to their m/z ratio
Only one of the separated ion beams is allowed to
strike the detector

The separation according to m/z ratio is based upon
the radio frequency & direct current voltages applied
to these electrodes

These voltages produce an oscillating electric field
that transmits ions according to their m/z value by
alternatively focusing them in different planes

Used mostly with MALDI

The time-of-flight (TOF) analyzer uses an electric
field to accelerate the ions through the
same potential, and then measures the time they take
to reach the detector

The smaller ions will reach the detector first because
they will acheive great velocities

The larger ions will have lesser velocities & reach the
detector late
The final element of the mass spectrometer is the
detector

The detector generates a signal current from
incident ions by generating secondary electrons
which are further amplified

Types:

Faradey Cup
Electron Multiplier
Photomultiplier Conversion Dynode

Concept: A change in charge on a metal plate results
in a flow of electrons
The flow creates a current

When a single ion strikes the surface of a dynode in
faradey cup, it results in ejection of several electrons

This ejection induces a current in the cup
Uses a series of dynodes maintained at successively
higher potentials

Thus,electrons released by the 1
st
dynode (when ion
impinges on it) are dragged to 2
nd
dynode because it
has a higher potential

Highly sensitive
Ions strike a dynode resulting in emission of
electrons

These electrons are made to strike a phosphorous
screen


The screen releases photons


Photons detected by a photomultipier

Complex mixtures are now analyzed without prior
purification by tandem MS

It employs the equivalent of 2 mass spectrometers
linked in series

The 1
st
spectrometer separates individual peptides
upon their differences in mass

By adjusting the field strength of 1
st
magnet, a single
peptide can be directed into 2
nd
mass spectrometer
,where fragments are generated and their mass
determined

Applications:

1) Identification & quantification of proteins

2) Drug screening

3) Pesticides & pollutants screening

4) Used to screen blood samples from new borns for the
presence & conc of proteins,F.A,other metabolites

5) Screening of inborn errors of metabolism (phenyl
ketonuria, ethylmalonic encephalopathy,glutaric acidemia
type 1)

Gas chromatography-mass spectrometry

In this technique, a gas chromatograph is used to separate
different compounds.

This stream of separated compounds is fed online into
the ion source, a metallic filament to which voltage is
applied.

This filament emits electrons which ionize the compounds.

The ions can then further fragment, yielding predictable
patterns.

Intact ions and fragments pass into the mass spectrometer's
analyzer and are eventually detected
Liquid chromatography-mass spectrometry

Separates compounds chromatographically before they are
introduced to the ion source and mass spectrometer.

It differs from GC/MS in that the mobile phase is liquid,
usually a mixture of water and organic solvents

Most commonly, an electrospray ionization source is used
in LC/MS. There are also some newly developed ionization
techniques like laser spray
PROTEIN CHARACTERIZATION:
Proteins are 1
st
digested into smaller peptides using
different proteases
A collection of these smaller peptides is then
introduced into the mass analyzer

ANALYSIS OF BIOLOGICAL NONCOVALENT
COMPLEXES
Electrospray ionization gets these noncovalent
complexes into gaseous phase & MS can be used to
observe these complexes
Eg: Hb complex , DNA duplex , cell surface
carbohydrates, whole viruses

CHARACTERIZAION OF SMALL BIOMOLECULES
ASSOCIATED WITH DIFFERENT BIOLOGICAL
STATES
MS successfully discovered that cis-9,10-octadecenoamide
was present in the sleep state & was absent during the
wake state

APPLICATIONS IN VIROLOGY:
Identification of a virus in a given sample by analyzing
the mass of the capsid proteins or DNA/RNA through MS

SEQUENCING PEPTIDES & OLIGONUCLEOTIDES
MALDI has been used recently to sequence proteins &
oligonucleotides
Isotope ratio MS: isotope dating and tracking
Mass spectrometry is also used to determine
the isotopic composition of elements within a sample
Trace gas analysis

selected ion flow tube (SIFT-MS), andproton
transfer reaction (PTR-MS), are variants
of chemical ionization dedicated for trace gas
analysis of air, breath or liquid headspace

Use well defined reaction time allowing
calculations of analyte concentrations from the
known reaction kinetics without the need for
internal standard or calibration.

Atom probe

An atom probe is an instrument that combines time-of-
flight mass spectrometry and field ion microscopy (FIM)
to map the location of individual atoms.


Pharmacokinetics

Pharmacokinetics is often studied using MS because of
the complex nature of the matrix (often blood or urine)
and the need for high sensitivity to observe low dose and
long time point data.

The most common instrumentation used in this
application is LC-MS with a triple quadrupole MS
Glycan analysis

Mass spectrometry (MS), with its low sample
requirement and high sensitivity, has been the
predominantly used in glycobiology for
characterization and elucidation of glycan structures.

Mass spectrometry provides a complementary
method to HPLC for the analysis of glycans
Space exploration
As a standard method for analysis, mass spectrometers
have reached other planets and moons.

Two were taken to Mars by the Viking program.

In early 2005 the Cassini-Huygens mission delivered a
specialized GC-MS instrument aboard the Huygens
probe through the atmosphere of Titan, the largest
moon of the planet Saturn.

This instrument analyzed atmospheric samples and
was able to vaporize and analyze samples of Titan's
frozen, hydrocarbon covered surface once the probe
had landed.
Advantages

Provides molecular weights of peptides and proteins
with high accuracy (0.1-0.01%)

Highly sensitive; requires fmol-pmol quantities of
sample

Sample purity not important

Can be coupled with on-line separation methods such
as HPLC and capillary electrophoresis for the analysis
of mixtures


Disadvantages

Noncovalent complexes are often disrupted

Cannot distinguish stereoisomers

Expensive instrumentation