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Mass Spectrometry

mass spectrometry, also called mass spectroscopy, analytic technique by which chemical substances are identified by the sorting of gaseous ions in electric and magnetic fields according to their mass-to-charge ratios.

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Ganesh V Gaonkar
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73 views

Mass Spectrometry

mass spectrometry, also called mass spectroscopy, analytic technique by which chemical substances are identified by the sorting of gaseous ions in electric and magnetic fields according to their mass-to-charge ratios.

Uploaded by

Ganesh V Gaonkar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Mass Spectrometry

How to investigate
small “things”?????
things that are so small that you can
barely or even not see them with your eyes
“things” that are looked
at and
have to be analyzed are
so small that they cannot
be seen
with the naked eye at all
and even a microscope or
magnifying glass will not
do the trick

can “weigh” them with a


special type of scale balance
The building blocks of matter
“Everything around us consists of very small building
blocks “atoms” and
Clusters of building blocks “molecules”
Atoms and molecules are so small that we cannot see them
The different building blocks of
different materials have a different mass
Where are mass spectrometers used?
Mass spectrometers are used in industry and academia for both
routine and research purposes.

The following list is just a brief summary of the major mass


spectrometric applications:
Pharmaceutical: drug discovery, combinatorial chemistry,
pharmacokinetics, drug metabolism
Clinical: neonatal screening, haemoglobin analysis, drug testing
Environmental: PAHs, PCBs, water quality, food contamination
Geological: oil composition
Biotechnology: the analysis of proteins, peptides,
oligonucleotides
How can mass spectrometry help biochemists?
Accurate molecular weight measurements: 
sample confirmation, to determine the purity of a sample, to verify
amino acid substitutions, to detect post-translational modifications, to
calculate the number of disulphide bridges
Reaction monitoring: 
to monitor enzyme reactions, chemical modification, protein digestion
Amino acid sequencing: 
sequence confirmation, de novo characterization of peptides,
identification of proteins by database searching with a sequence "tag"
from a proteolytic fragment
Oligonucleotide sequencing: 
the characterization or quality control of oligonucleotides
Protein structure: 
protein folding monitored by H/D exchange, protein-ligand complex
formation under physiological conditions, macromolecular structure
determination
Mass Spectrometry
as a tool for Biomolecule Characterization
Mass Spectroscopy

• Effective method for determining the weight and


structure of compounds.
• Separates ions on the basis of mass to charge ratio
• Only picomolar concentrations required
• It is advantageous to other methods of analysis in its
ability to separate and analyze mixtures of compounds.
• It is an extremely sensitive technique with current
levels of accuracy below one mass unit.
Mass Spectroscopy
• Analogy of smashing a vase with a
hammer, and determining what the
vase looked like by putting the pieces
back together
• Difficulty is that all bonds can
potentially break, but not all bonds
break equally
+
ABC
ABC + +
AB C
+ +
A BC
Important
Mass Spectroscopy Terms
• Molecular ion- peak where molecule is not broken,
just lost an e– (ABC+ in this figure)
• Fragment ions- all other ions
• Base peak- most abundant peak in spectrum (a
fragment ion)

+
ABC
ABC + +
AB C
+ +
A BC
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
General Setup
Common to all mass spectrometers are
(1) Sample Inlet (2) Ionization Source
(3) Mass Analyzer (4) Ion Detector
(5) Vacuum System
Ionization
• Either removal of e- from the
molecule (cation), or adding an
e- (to form anion)
• Electron removal more easier
and efficient, hence positive ion
mass spectroscopy is more
common
• Removal of protons (H) also
produce ions
• Adduct formation with NH4+ or
CH5+ can produce ions
(chemical ionization)
Ionization Methods
• Electron Impact (EI - Hard method)
– small molecules, 1-1000 Daltons, structure

• Fast Atom Bombardment (FAB – Semi-hard)


– peptides, sugars, up to 6000 Daltons

• Electrospray Ionization (ESI - Soft)


– peptides, proteins, up to 200,000 Daltons

• Matrix Assisted Laser Desorption (MALDI-Soft)


– peptides, proteins, DNA, up to 500 kD
Electron Impact Ionization (EI)

• The sample must be delivered as a gas which is usually


accomplished by heating the sample to vaporize it off of the probe.
• Once in the gas phase, the compound passes into an electron
ionization region where it interacts with a beam of electrons of
nearly homogeneous energy (70 electron volts), typically causing
electron ejection and some degree of fragmentation
• Electron source- filaments of metals like rhenium or tungsten
heated to 2000K emit e- which are accelerated by a potential
gradient
EI is not for proteomics
 EI shatters chemical bonds
 A peptide/protein contains (20 different) amino
acids
 EI would shatter the peptide not only into amino
acids but also amino acid sub-fragments
 Result is tens of thousands of different signals from
a single peptide — too complex to interpret
 EI might be well suited for some applications in
metabolomics
Electrospray Ionization (ESI)
Electrospray ionization generates
ions directly from solution by
creating a fine spray of highly
charged droplets in the presence
of a strong electric field.

• Sample dissolved in polar, volatile buffer (no salts) and


pumped through a stainless steel capillary (70 - 150 mm) at a
rate of 10-100 mL/min
• Strong voltage (3-4 kV) applied at tip along with flow of
nebulizing gas causes the sample to “nebulize” or aerosolize
• Aerosol is directed through regions of higher vacuum until
droplets evaporate to near atomic size (still carrying charges)
Electrospray Ionization (ESI)

• Strongly affected by salts and detergents

• Positive ion mode measures (M + H)+


(add formic acid to solvent)

• Negative ion mode measures (M – H) –


(add ammonia to solvent)
Positive or negative mode
Functional groups that readily accept H + (such as
amide and amino groups found in peptides and
proteins) can be ionized using positive mode ESI

Functional groups that readily lose a proton (such


as carboxylic acids and hydroxyls as found in
nucleic acids and sugars) should be ionized using
negative mode ESI
Matrix-Assisted Laser Desorption/
Ionization (MALDI)

• Sample is ionized by bombarding sample


with laser light
• Sample is mixed with a UV absorbent
matrix
• Light wavelength matches that of
absorbance maximum of matrix so that the
matrix transfers some of its energy to the
analyte (leads to ion sputtering)
Matrix-Assisted Laser Desorption/
Ionization (MALDI)

In MALDI analysis, the analyte is first


co-crystallized with a large molar
excess of a matrix compound, after
which pulse UV laser radiation of this
analyte-matrix mixture results in the
vaporization of the matrix which
carries the analyte with it.

The ability to generate MALDI-MS data on whole proteins and


proteolytic fragments is extremely useful for protein
identification and characterization.
Matrix

• Matrix - crystallized Biomolecules


– 3,5-dimethoxy-4-hydroxycinnamic acid sinapinic acid),
 -cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix)
– 2,5-dihydroxybenzoic acid(DHB)

• Solution of one of these molecules is made, in a mixture of highly purified


water and an organic compound (normally acetonitrile (ACN) or ethanol).
Normally some trifluoracetate (TFA) is also added.

• Good example of a matrix-solution: 20 mg/mL sinapinic acid in


ACN:water:TFA (50:50:0.1).
Methods of Separation- Analysers
Different types of Mass Analyzers differ in the physical
properties they manipulate.
• Magnetic, which depends on Voltage
• Time of Flight, which depends on Mass
• Electric Quadrupole, which depends on AC/DC Currents
• Ion Trap, similar to quadrupole
Common to these Mass Analyzers is the separation of particles
on the basis of mass to charge ratios (m/z).
Time of Flight Analyser
• Separates ions based on
flight time
• Operates in pulsed mode
• Ions accelerated by an
electric field
• Lighter ions reach the
detector first
L in e a r Tim e O f F lig h t tu b e

io n so u rc e

d e te cto r

tim e o f flig h t

R e fle c to r Tim e O f F lig h t tu b e

io n so u rc e

d e te cto r
re fle c to r

tim e o f flig h t

• Reflectron TOF-MS: Improved mass resolution in MALDI TOF-MS obtained by


utilization of a single-stage or a dual-stage reflectron (RETOF-MS).
• Reflectron, located at the end of the flight tube, used to compensate for the
difference in flight times of the same m/z ions of slightly different kinetic energies
by means of an ion reflector.
• Results in focusing the ion packets in space and time at the detector
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