this is to know about mass spectrometry technique which mainly used in proteomiics

2. Presented by…
SHAILESH BHARTI
B.TECH (BIOTECH)
Presented to…
DR.AKASH TOMAR
Deptt. of(RDT)

3. Matrix assisted laser desorption/ionization-Time
of flight.

4. 1. Mass is how heavy something is without gravity.
2. Another way of describing mass is:
mass is how much matter an object has
3.Mass is measured in grams (g) or kilograms (kg).

5. What is spectrometer?
In physics, a spectrometer is an apparatus to
measure a spectrum. Generally, a spectrum is a graph
that shows intensity as a function of wavelength, of
frequency, of energy, of momentum, or of mass.
The branch of science concerned with the investigation and
measurement of spectra produced when matter interacts with or
emits electromagnetic radiation.
What is spectroscopy?

7. About mass spectrometry
• Mass spectrometry involves the ionization of target
molecules in a vacuum, and accurate measurement
of the mass of the resulting ions.
• The technique is a powerful qualitative and quantitative
tool, routine analyses are performed down to the
femtogram (10-15 g) level and as low as the zeptomole
(10-21 mol) level for proteins.

8. General Schematic
A mass spectrometer needs to perform three functions:
• Creation of ions – the sample molecules are subjected to a high
energy beam of electrons, converting some of them to ions.
• Separation of ions – as they are accelerated in an electric field,
the ions are separated according to mass-to-charge ratio (m/z).
• Detection of ions – as each separated population of ions is
generated, the spectrometer needs to qualify and quantify them.
2. The differences in mass spectrometer types are in the
different means to carry out these three functions.
3. Common to all is the need for very high vacuum (~ 10-6
torr), while still allowing the introduction of the sample.

9. - Separates gas phase ionized atoms, molecules, and
fragments of molecules
- Separation is based on the difference in mass-to-charge ratio (m/z)
m = unified atomic mass units (u)
1 dalton (Da) = 1 u = 1.665402 x 10-27 kg
z = charge on the ion (may be positive or negative).
- Computer algorithms is used for correlating MS-determined
mass with virtual mass derived from protein database.

10. Mainly 3 components parts:-
1. Ionizer :- which converts the analyte into gas phase ions.
2. Mass analyzer :- which separates the ions according to their
mass/charge(m/z).
3. Ion detector

11. Creating ions
1. Hard ionization and soft ionization
2. Inductively coupled plasma
3. Other ionization techniques
Mass selection
1. Sector instruments
2. Time of flight
3. Quadrupole mass filter
4. Ion traps.
5.Fourier transform ion cyclotron resonance
Types of MS on different basis:-

12. In proteomics we use mainly two types of mass spectrometers
1. Electrospray ionization (ESI)
2. MATLDI- TOF
Tandem mass spectrometer (MS/MS) is used to sequence of small
peptides (>25) residues .

13. ELECTROSPRAY IONIZATION(ESI)
Discovered by:-John Fenn

14. Peptide is sprayed from a narrow capillary tube maintained at
high voltage (4000 V), forming fine,highly charged droplets
from which the solvent rapidly evaporates.
This yields a series of gas phase macromolecular ions that
typically have ionic charges in the range 0.5 to 2 per
kilodalton.

15. 2.Matrix-assisted laser desorption/ionization (MALDI)

17. Matrix-assisted laser desorption/ionization-Time Of
Flight(MALDI-TOF).
Soft ionization:- The ionization of large molecules, such as
proteins and nucleic acids, without causing
significant amounts of fragmentation.
A soft ionization method used for peptide mass
fingerprinting.
Time of flight (TOF)
A mass analyzer that determines the mass : charge
ratio of an ion by measuring the time taken by ions to
travel down a flight tube to the detector (Karas &
Hillenkamp1988).
The term (MALDI) was coined in 1985 by Franz Hillenkamp,
Michael Karas and their colleagues.

18. Protein spots are excised from a 2D gel and digested with a specific endopeptidase,
such as trypsin, to generate peptide fragments.
Steps of MALDI -TOF
The analyte, a mixture of peptide fragments resulting from tryptic digestion
of a particular protein, is first mixed with a light-absorbing “matrix
compound” such as dihydroxybenzoic acid, in an organic solvent.
The solvent is then evaporated to form crystals and these are transferred to a vacuum.
The laser energy is absorbed and then emitted (desorbed) as heat, resulting in expansion of
the matrix and anylate into the gas phase.
A high voltage is applied across the sample to ionize it, and the ions are accelerated towards
the detector.
Analysis of mass spectrum and determination of mass

19. UV MALDI Matrix List
Compound Other
Names
Solvent Wavelength
(nm)
Applications
2,5dihydroxy
benzoic acid
DHB,
Gentisic acid
acetonitrile,
water,
methanol,
acetone,
chloroform
337, 355,
266
peptides,nucleotide,
oligonucleotides,
oligosaccharides.
3,5dimethoxy4
hydroxycinnam-
ic acid
Sinapic acid;
Sinapinic acid;
SA
acetonitrile,
water,
acetone,
chloroform
337, 355,
266
peptides,
proteins, lipids
4hydroxy3
methoxycinnaic
acid
Ferulic acid acetonitrile,
water,
propanol
337, 355,
266
Proteins
αCyano4hydroy
cinnamic acid.
CHCA acetonitrile,
water,
ethanol,
acetone
337, 355 peptides, lipids,
nucleotides
Picolinic acid PA Ethanol 266 oligonucleotids

20. Examples of Lasers used
- IR laser: CO2 laser
- UV laser: Nd:YAG (neodymium-doped yttrium
aluminium garnet; Nd:Y3Al5O12) is a crystal
that is used as a lasing medium for solid-
state lasers.)

21. THE MASS SPECTRUM
- A plot of relative abundance vs m/z
- The most abundant peak is known as the base peak
- The base peak is scaled to 100
- Spectrum shows fragmentation patterns
- The m/z values and the fragmentation pattern are used to determine
the molecular weight and structure of organic compounds
- Provides the accurate mass of a given isotope not the
weighted average.

23. RESOLVING POWER
- The ability of a mass spectrometer to separate ions of two
different m/z values
- Resolving power = M/∆M
- M = mass of one singly charged ion
- ∆M = difference in mass between M and the next m/z value
- The resolving power of ions in the 600 range = 600
- The resolving power of ions in the 1200 range = 1200

24. - Gas phase molecules are ionized by a beam of high energy
electrons
- Electrons may be ejected from molecules (ionization) or bonds
in molecules may rapture (fragmentation)
- Ions are then accelerated in a field (sector) at a voltage V
- Sector can have any apex angle (60o and 90o are common)
- Most modern instruments combine both electric sector
and magnetic sector (double-focusing MS)
MAGNETIC SECTOR MASS ANALYZER

25. MAGNETIC SECTOR MASS ANALYZER
- The electric sector acts as an energy filter
- m/z range is 1 – 1400 for single-focusing and
5,000 – 10,000 for double-focusing instruments
- Energy of each ion = zV
- Kinetic energy depends on charge and voltage but not
on mass of ion
- Ions with small masses must travel at a higher velocity
than ions with larger masses

26. MAGNETIC SECTOR MASS ANALYZER
- For single positively charged ions
zVmv
2
1 2

1/2
m
2zV
v 






m = mass of ion
v = velocity of ion
z = charge of ion
V = accelerating voltage
- V changes as m varies such that ½ mv2 is
constant
m
1
αv

27. - Ions enter a curved section of a homogeneous magnetic
field B after acceleration
- Ions move in a circle with radius r
- Attractive force on magnet = Bzv
- Centrifugal force on the ion = mv2/r
- The two forces are equal if the ion follows the radius of
curvature of the magnet
MAGNETIC SECTOR MASS ANALYZER

28. MAGNETIC SECTOR MASS ANALYZER
Bzv
r
mv 2

Bz
mv
r 
Substituting for v and rearranging gives
2V
rB
z
m 22


29. MAGNETIC SECTOR MASS ANALYZER
- Radius of circular path depends on m/z if V and B are kept constant
- Ions with different m/z travel in circles with different radii
- Basis of separation by m/z
- Ions with the right m/z reach the detector and others hit the
sides of the instrument and be lost
- Which m/z to reach the detector can be selected by varying V or B
- B is varied and V is kept constant in modern instruments

30. TIME OF FLIGHT (TOF) ANALYZER
- Makes use of a drift tube
- Pulses of ions are accelerated into an evacuated drift tube
(free of field or external force)
- Velocity of an ion depends on m/z
(depends on mass if all ions have the same charge)
- Lighter ions move faster along the tube than heavier ions
- Ions are separated in the drift tube according to their velocities (v)

31. 1/2
m
2zV
v 






TIME OF FLIGHT (TOF) ANALYZER
- V = accelerating voltage
- If L is the length of tube (typically 1-2 m) and t is the flight
time of ion, then v = L/t
- Implies mass-to-charge ratio and flight time can be found from.
2
2
L
2Vt
z
m

2zV
m
Lt 
- An ion mirror called a reflectron is used to increase resolution.

32. 3. Tandem mass spectrometer (MS/MS )

33. two mass spectrometers coupled in series
:- The first mass spectrometer functions to select the peptide ion
of interest from other peptide ions as well as any contaminants
that may be present.
:- The selected peptide ion (P3) is then passed into a collision cell,
where it collides with chemically inert atoms such as helium. The
energy thereby imparted to a peptide ion causes it to fragment
predominantly at only one of its several peptide bonds, yielding
one or two charged fragments.
The molecular masses of the charged fragments are then
determined by the second mass spectrometer.

34. Applications of MALDI-TOF
Biochemistry
In proteomics, MALDI is used for identification of proteins isolated by using gel
electrophoresis: SDS-PAGE,size exclusion chromatography, affinity chromatography,
strong/weak ion exchange, isotope coded protein labelling (ICPL),and two dimensional
gel electrophoresis.
Organic chemistry
It allow chemists to rapidly analyze the results of Some synthetic macromolecules,
such as catenanes and rotaxanes, dendrimers and hyperbranched polymers and verify
their results.
Polymer chemistry
In polymer chemistry MALDI can be used to determine the molar mass distribution.

35. Microbiology
Identification of microorganisms such as bacteria or fungi.
Medicine
MALDI/TOF spectra are often utilized in tandem with other
analysis and spectroscopy techniques in the diagnosis of diseases.

36. Companies who manufactures the MALDI TOF instruments
There are 5 companies and 16 products of MALDI TOF
1.AB SCIEX (2)
2. Biomérieux (1)
3.Bruker Daltonics (6)
4. Shimadzu Europa (4)
5. Waters (3)

37. Products of Shimadzu
1. AXIMA ASSURANCE
2. AXIMA CONFIDENCE
3. AXIMA PERFORMANCE
4. AXIMA RESONANCE

38. TOF/TOF 5800 System
A product of AB SCIEX

39. Products of Bruker Daltonics
1. Microflex
2. Autoflex
3. MALDI pharma plus
4. New ultraflex Xtreme
5. rapifleX MALDI tissuetyper
6. Proteineer fc