MASS SPECTROMETRY

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MASS SPECTROMETRY

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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INTRODUCTION

• 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

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PRINCIPLE

• 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

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• 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

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INSTRUMENTATION

• 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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How does a Mass Spectrometer work?

• 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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Sample introduction

• 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

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Ionization

• Ionization means placing a charge on a neutral
  molecule
• Methods
• 1) Electron ionization
• 2) Electrospray
• 3) Matrix-assisted laser desorption/
  ionization(MALDI)

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Electron ionization

• 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

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Electrospray

• 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

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• 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

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Matrix-assisted laser desorption/ionization(MALDI)

• 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

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• 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

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Mass analyzers

• 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

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Magnetic sector mass analyzer

• J.J Thompson,who built the 1st 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

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• 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

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The quadrapole analyzer

• 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

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• 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

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Time of flight Analyzer

• 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

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DETECTOR

• 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

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Faradey Cup

• 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

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Ejection of several electronscurrent in
the cup
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Electron multiplier

• Uses a series of dynodes maintained at
  successively higher potentials
• Thus,electrons released by the 1st dynode (when
  ion impinges on it) are dragged to 2nd dynode
  because it has a higher potential
• Highly sensitive

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Photomultiplier Conversion Dynode

• 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

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TANDEM MS

• Complex mixtures are now analyzed without prior
  purification by tandem MS
• It employs the equivalent of 2 mass spectrometers
  linked in series
• The 1st spectrometer separates individual
  peptides upon their differences in mass
• By adjusting the field strength of 1st magnet, a
  single peptide can be directed into 2nd mass
  spectrometer ,where fragments are generated and
  their mass determined

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• 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)

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•  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

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• 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

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APPLICATIONS

• PROTEIN CHARACTERIZATION
• Proteins are 1st 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

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• 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

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• Isotope ratio MS isotope dating and tracking
• Mass spectrometry is also used to determine
  the isotopic composition of elements within a
  sample

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• 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.

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• 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

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• 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

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• 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.

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• 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

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• Disadvantages
• Noncovalent complexes are often disrupted
• Cannot distinguish stereoisomers
• Expensive instrumentation

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Thank you