Presented by…SHAILESH BHARTI B.TECH (BIOTECH)

Presented to…DR.AKASH TOMARDeptt. of(RDT)

MASS SPECTROMETRY

(MALDI-TOF)

Matrix assisted laser desorption/ionization-Time of flight.

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).

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?

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.

General SchematicA 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.

- 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).

PRINCIPLE

- Computer algorithms is used for correlating MS-determined mass with virtual mass derived from protein database.

Components 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

Creating ions

1. Hard ionization and soft ionization2. Inductively coupled plasma3. Other ionization techniques

Mass selection1. Sector instruments2. Time of flight3. Quadrupole mass filter4. Ion traps.5.Fourier transform ion cyclotron resonance

Types of MS on different basis:-

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 .

ELECTROSPRAY IONIZATION(ESI)

Discovered by:-John Fenn

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.

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

Matrix-assisted laser desorption/ionization-Time Of Flight(MALDI-TOF).

Soft ionization:- The ionization of large molecules, such asproteins and nucleic acids, without causingsignificant 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.

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

UV MALDI Matrix ListCompound Other

NamesSolvent Wavelength

(nm)Applications

2,5dihydroxybenzoic acid

DHB,Gentisic acid

acetonitrile,water,methanol,acetone,chloroform

337, 355,266

peptides,nucleotide,oligonucleotides,oligosaccharides.

3,5dimethoxy4hydroxycinnam-ic acid

Sinapic acid;Sinapinic acid; SA

acetonitrile,water,acetone,chloroform

337, 355,266

peptides,proteins, lipids

4hydroxy3methoxycinnaicacid

Ferulic acid acetonitrile,water,propanol

337, 355,266

Proteins

αCyano4hydroycinnamic acid.

CHCA acetonitrile,water,ethanol,acetone

337, 355 peptides, lipids,nucleotides

Picolinic acid PA Ethanol 266 oligonucleotids

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.)

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.

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

- 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

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

MAGNETIC SECTOR MASS ANALYZER

- For single positively charged ions

zVmv21 2

1/2

m2zVv

m = mass of ionv = velocity of ionz = charge of ion

V = accelerating voltage

- V changes as m varies such that ½ mv2 is constant

m1αv

- 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

MAGNETIC SECTOR MASS ANALYZER

Bzvr

mv2

Bzmvr

Substituting for v and rearranging gives

2VrB

zm 22

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

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)

1/2

m2zVv

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

L2Vt

zm

2zVmLt

- An ion mirror called a reflectron is used to increase resolution.

3. Tandem mass spectrometer (MS/MS )

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. Theenergy thereby imparted to a peptide ion causes it to fragmentpredominantly at only one of its several peptide bonds, yielding one or two charged fragments.

The molecular masses of the charged fragments are thendetermined by the second mass spectrometer.

Applications of MALDI-TOFBiochemistry

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 dimensionalgel 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.

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.

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)

Products of Shimadzu

1. AXIMA ASSURANCE

2. AXIMA CONFIDENCE

3. AXIMA PERFORMANCE

4. AXIMA RESONANCE

TOF/TOF 5800 System

A product of AB SCIEX

Products of Bruker Daltonics

1. Microflex2. Autoflex 3. MALDI pharma plus4. New ultraflex Xtreme5. rapifleX MALDI tissuetyper6. Proteineer fc

QUESTIONS .?????If any…..