Mass spectrometry is an analytical technique for measuring the mass of biological molecules by converting them into gaseous ions, which are then separated and detected based on their mass-to-charge ratio. The document outlines the components, types of ion sources, mass analyzers, and the methodology behind MALDI-TOF mass spectrometry, detailing the production and detection of ions through various techniques. Applications of mass spectrometry span across biochemistry, organic chemistry, microbiology, and medicine.

1. MASS SPECTROMETRY
&
MALDI-TOF
By
Mahek Sharan
Mtech Biotechnology
India

2. Contents
• Introduction
• Basic steps
• Basic components
• Types of vacuum pumps
• Types of ion sources
• Types of mass analyser
• MALDI TOF
1. Matrix
2. Laser
3. Principle of mechanism
4. TOF
5. Detector
• Applications

3. MASS SPECTROMETRY
• The analytical technique by which the biological molecules are firstly
converted into gaseous ions and then are separated by the mass to charge
(m/z) ratio and detected
• This technique could accurately measure the mass of the low as well as high
molecular compound by 2 techniques
1. ESI
2. MALDI-TOF

4. The basic steps involved
1. Production of ions in gaseous phase
2. Acceleration of ions in the applied electric field at specific velocity
3. Separation of the ion in the mass analyser
4. Detection by m/z ratio by the detectors

5. Basic component of mass spectrometry
High vacuum system

6. Components
• High vacuum system
3 types of pumps used
1. Turbomolecular pump
2. Diffusion pump
3. Rotatory vane pump
• Sample inlet
• Many types of sample or target plate are obtained by
1. HPLC
2. Gas Chromatography
3. Capillary Electrophoresis
4. Solid probe
5. Direct chemical
• Ion source
1. Electron impact ionisation
2. Chemical ionisation
3. Electrospray Ionisation
4. Fast atom bombardment
5. MALDI
• Mass analyser
1. TOF
2. Quadrupole
3. Ion cyclotron
4. Ion trap
5. Magnetic sector
• Detector
1. Conversion diode
2. Electron multiplier
3. Microchannel plate
4. Array detector

7. Types of Vacuum pump
• Turbomolecular pump
• Uses the principle of momentum of air particles
can be given and changed when it is collided by
the solid surface
• There are rotots hitting gas molecules and
making them leave from exhaust by the help of
Backing pump and hevlovk pump for lowe
pressure generation
• Diffusion pump
• Uses high speed jet of vapours to direct
molecules out of the chamber
• Oil used are silicone oil or polyphenyl oil
• High level vacuum created

8. • Rotary vane pump
• Uses vane fixed on rotors to make gaseous
molecules leave the chamber
• It traps the air and later move it out by the
rotation in the cavity
• This displacement pump is less suitable for high
viscous liquids

9. Types of Ion sources
• Electron Impact Ionisation (EI)
• The metal plate / filamnet is heated in a vacuum
system resulting in the positive charge of the
sample in gaseous phase by the electrons
released in a form of beam colliding to it
• Fragmentation of the ions occur into radical and
ions, called Molecular ions and radicals are of
no use only true ions, called Product ions
ionise the samples
• Chemical Ionisation (CI)
• The ion source is the chemical involved
Methane and Ammonia which are carrying the
charge and can either add or take an electron to
make the sample ions charged
• The charging of the samples done by these two
chemicals serves the role in high vacuum
chambers

10. • Electrospray Ionisation (ESI)
• It is a soft ionisation technique (Liquid secondary
ionisation)
• Uses jet spray from a capillary nozzle to ionise the
sample ions by aerosol conversion in presence of
inert gases like nitrogen or in vacuum
• The spray liquids used are the water mixed organic
acids like methanol, acetonitrile
• The liquid turns into vapours and undergo fission
and leaving analyte ions with charge causing
sample molecules to charge
• Fast atom bombardment (FAB)
• This is a soft ionisation technique (secondary ion)
• It utilises the inert gases like Argon, Neon to ionise
the sample molecules by colliding the inert gases
with fast moving electrons
• These fast atom beam when collided with the
matrix (glycerol) mixed sample ions generate the
ions with charge by fragmentation of the molecule

11. Types of Mass analyser
• Quadrupole
• The separation of the ions according to the mass
and charge ratio by the 4 parallel hollow
cylindrical rods
• The rods have a particular potential applied to it
as the potential changes, the ions of the sample
separated according to it
• The ions having a particular trajectory will only
reach the detector and rest will collide to the rod
• Ion trap mass analyser
• Ions are separated according to two fields
applied : Magnetic and electrical
• The magnetic filed surrounding the ions make
the mass of the ions equal and then the potential
varied could be used for the separation of
molecules only on the basis of the charge and
vice versa
• The ions are trapped inside the electrodes of
two types with similar radio frequency applied

12. • Magnetic Sector
• The analyser uses the magnetic field to separate
out the ions
• The magnetic places in between the ion beam
and detector gives the ion their own trajectories
based on their m/z ratios
• The particular m/z ratio ions are separated out
by this
• Plasma desorption Mass analyser
• The plasma (nuclei without electrons in orbital)
is mixed with matrix in solid state
• A californium atom which undergoes fission to
form Ba and Tf and the two atoms move
discretely in collinear opposite direction
• One in opposite of sample triggers a clock for
start of detector and other triggers the ions to
move to TOF analyser from sample foil for
separation

13. Ion source and Mass analyser:
MALDI-TOF
• Here the ion source is MALDI (Matrix Assisted Laser Desorption and
Ionisation) the mass analyser is TOF

14. • Matrix used in MALDI-TOF

15. • Types of Laser used
Lasers
Nitrogen
Laser
337nm
Nd: YAG
355:266nm

16. Principle of mechanism
• The laser is fired at the matrix crystals in the dried-droplet spot.
• The matrix absorbs the laser energy. The hot plume produced during ablation contains many species:
1. neutral and ionized matrix molecules,
2. protonated and deprotonated matrix molecules,
3. matrix clusters and nanodroplets.
• The matrix is then thought to transfer protons to the analyte molecules, thus charging the analyte.
• An ion observed after this process will consist of the initial neutral molecule [M] with ions added or removed.
• MALDI is capable of creating singly charged ions or multiply charged ions ([M+nH]n+) depending on the
nature of the matrix, the laser intensity, and/or the voltage used.
• The ions are then transferred to the TOF analyser

17. TOF
• TOF- Time of flight mass analyser
• The type of a mass spectrometer most widely used with MALDI due to its large mass
range.
• MALDI-TOF instruments are often equipped with a reflectron that reflects ions using
an electric field. This increases the ion flight path, thereby increasing time of flight
between ions of different m/z and increasing resolution.
• MALDI has been coupled with IMS-TOF MS to identify phosphorylated and non-
phosphorylated peptides.
• Time of flight thus separates according to the m/z ratio as the lightest ion reaches first
to the detector and heaviest in the last

18. Detectors
• The final element of the mass spectrometer is the detector
• The detector records either the charge induced or the current produced when an ion
passes by or hits a surface.
• The detector used commonly is electron multiplier is used, though other detectors
including Faraday cups and ion-to-photon detectors are also used.
• The number of ions leaving the mass analyzer at a particular instant is typically
quite small, considerable amplification is often necessary to get a signal.
• Microchannel plate detectors are commonly used in modern commercial

19. Tandem mass spectrometry
• A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by
some form of molecule fragmentation
• Here, one mass analyzer can isolate one peptide from many entering a mass spectrometer.
• A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to
fragment by collision-induced dissociation (CID).
• A third mass analyzer then sorts the fragments produced from the peptides.
• Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap.
• There are various methods for fragmenting molecules for tandem MS:
• Electron capture dissociation (ECD),
• Electron transfer dissociation (ETD),
• Infrared multiphoton dissociation (IRMPD), etc.

20. APPLICATIONS
• Biochemistry
• Polymer chemistry
• Organic chemistry
• Microbiology
• Parasitology
• Medicine
• Protein complexes detection