MALDI - TOF

HIMA HARIDASAN

INTRODUCTION•Proteome – complete set of proteins encoded in a

genome

•Proteomics

•Protein identification requires their separation by methods :_

1. IEF2. SDS- gel electrophoresis3. Mass spectroscopy (MS)

MASS SPECTROSCOPY

•Separation of charged molecules/ molecules according to their mass to charge

•Determines relative molecular mass (Mr)

•High resolution, precision, & sensitivity

•Requires charged gaseous molecules for its analysis

COMPONENTS OF MASS SPECTROSCOPY

• Ionization source 1. Matrix assisted laser desorption/ ionization – time of

flight(MALDI)2. Electrospray (ES)

• Analyser m/z : TOF, Magnet,..

• Detector : Photomultiplier, Electron multiplier, ..

COMPONENTS OF MASS SPECTROSCOPY

Inlet Detector DataSystem

MassAnalyzer

High Vacuum System

IonSource

Time of flight (TOF)QuadraupoleIon TrapMagnetic SectorFTMS

Turbo molecular pumps

HPLCFlow injectionSample plate

Microchannel PlateElectron MultiplierHybrid with photomultiplier

PCSun SPARK StationDEC Station

MALDI - TOF• Synonyms : MALDI, TOF – MS

•MALDI- Term was coined in 1985 by Franz Hillenkamp, Michael Karas

• Principle : velocity of ions depend on its mass & energy, the time taken by an ion to travel a specified distance (TOF)

•Use : MALDI ionization & TOF of the ions to reach the detector as a parameter to measure m/z ratio.

• Sample excitation – from the energy of a laser transferred via a UV light absorbing matrix

MATRIX

•Conjugated organic compound or weak organic acid

•Weak organic acid – derivative of cinnamic acid &

dihydroxybenzoic acid

• Eg : sinapinic acid- proteins (10Da)

•Usually mixed with sample

•Absorbs maximum light at a wavelength (λ) of the laser,

typically a N2 laser of 337nm or yytrium – aluminium garnet

(Nd-YAG) AT 335nm.

•Acts as an absorbing media for the UV light

WORKINGSample

(1 – 10 pmolmm-3) Mixing Excess matrix

Sample – matrix

Dried on target plate

Co- crystallization UV rays

Desorption Pulses of laser light

Rapid excitation

Rapid heating of the region

Matrix & analyte ions ejection Into

Gas phase Results in

Explotion of sample region into high vacuum

Gas phase protonated molecules Enter

TOF

Sample molecule ionization

MH+ + A - > M + AH+

(M - H)- + A - > [A – H-] + M

MALDI –IONIZATION MECHANISM

SAMPLE CONCENTRATION FOR MATRIX

•Maximum sensitivity if samples a diluted to a specific

concentration range

• For unknown sample concentration a dilution series may be

needed

• Sample loaded as a spot

• Peptides & proteins give best spectra (10pmolmm-3)

• Especially glycoproteins (10pmolmm-3)

•Oligonucleotides- better spectra at 10 - 100pmolmm-3

• Polymers - 100pmolmm-3

MALDI ADVANTAGES

•Gentle Ionization technique

•High molecular weight analyte can be ionized

•Molecule need not be volatile

•Sub-picomole sensitivity easy to obtain

•Wide array of matrices

TOF• Best type of mass analyser to couple to MALDI

• Unlimited mass range

• Macromolecules of Mr > 400,000 – accurately measured

• 100 pulses of laser light (10nanoseconds)

• Result – seen as a good spectrum

• Camera – tracks the laser beam around the MALDI

TOF ADVANTAGES

•All ions detected at once

•High mass accuracy and resolving power possible

•Reasonable performance for cost▫<5 ppm mass accuracy and >20,000 resolving power

commercially available

•High mass, low charge ions not a problem▫Theoretically unlimited mass range

• Principle:-Ions

EnterFlight tube

ReachesDetector

Acceleration of ionsFixed point & initial time

Ion separation(according to m/z ratio)

TOF Converted to

Mass

LINEAR TOF MS

•Uses MALDI & TOF

•Samples are deposited on a metal substrate (100)

Analyte spots laser

Short burst of ions

Ion acceleration to a fixed KE

Ions travel down a flight tube

DE MALDI – TOF MS

•Delayed extraction TOF MS

•Mass dependent

•Used for DNA sequencing

•Lacks resolution & sentivity to analyze complex mixtures

RE TOF MS

•Reflectron TOF MS

•Single stage or dual stage reflectron – at the end of flight tube

•High resolution

•Reflectron – compensate for the difference in the TOF of ions with same m/z ratio, but same KE

DELAYED EXTRACTION(DE)

• Extraction of ions generated by a high electrostatic field occurs

• Ions from different spots have different energy

• Energy spreads, so broadens the peak corresponding to each

ion (low accuracy)

• If extraction is delayed until all ions are formed, spread

minimizes (high accuracy) : DE

• Here extraction occurs by high voltage.

• Lengthening time of delay controls ion fragmentation degree.

POST SOURCE DECAY• Ion extraction results in ion fragmentation

•Biological molecules gives rise to molecules that have been extracted before this dissociation is complete

•Fragment ion’s velocity = precursor velocity

•Use of reflector overcomes this.

•Give structural information

MALDI – TOF INSTRUMENT COMPONENTS

• Sample target plate

• High vacuum chamber

• Camera

• Laser beam source

• Clock

•Flight tube

•Deflector

•Data system

•Reflectron

Camera

Laser

Sample plate

Pumping Pumping

Timed ion selector Reflector

Linear detector

Extractiongrids Reflector

detector

WORKING OF THE COMPONENTS

Sample-matrix

Target plate

High vacuum chamber

Camera tracks laser beam

Irradiation with laser pulses

Clock (on)

Measures TOF EF

Acceleration of ions to same kinetic energy

Ions fly through the flight tube

Ion separation (mass)

Ions strike detector

Data system (controls instrument parameters, acquires signal v/s time &

process the data )

TYPES OF MALDI SAMPLE PLATES

1. 100 well stainless steel flat plates

2. Four – hundred – spot Teflon – coated plates

3. Gold - coated plates

DETECTORS• Ions from MS impinge on its surface•Has a neutral charge on surface

• I flows through its surface • I gets amplified

• I gets converted to signal•Signal processed by a computer

•TIC – I 1 + I2 + ……..In

•TIC – to measure during online MS

REFLECTRON

•Reflector•An ion mirror that provides higher resolution

• Increases overall path length for an ion •Corrects minor variation in the energy spread of ionsof the same mass.

•Has a gradient electric field •The depth to which ions will penetrate this field,

before reversal of direction of travel, depends upon their energy.

•Higher energy ions travels more & vice versa. •Thus, TOF gets focussed

•Neutral fragments - unaffected by deflection• Improves resolution & mass accuracy

•Allows structure & sequence information collection by PSD analysis.

•Focuses charged fragments of a specific range of m/z

•So, a number of spectra are run at different settings & stiched together to generate composite spectrum.

APPLICATIONS

Chemistry

Pharmaceutical analysis

Clinical analysisTaxonomy

MICROBIOLOGY

CONCLUSION

•MALDI – TOF

•Working

•Measures only mass

• Instrument components

•Working of the instruments

REFERENCES

•Ashim k. Chakravarty - Intoduction to Biotechnology – 2013- Oxford university press – New Delhi – Pg. no : 301

•Glazer N. Alexander, Hiroshi Nikaido – Fundamentals of Applied Microbiology – second edition – 2008 – Cambridge university press - Pg. no : 159 – 162

•H,K. Das – Textbook of Biotechnology – fourth edition - 2011- Beekem printers – NewDelhi - Pg. no : 135

•H.S. Chawla – Introduction to Plant Biotechnology – third edition – 2013 – Oxford IBH Publishing company pvt ltd – NewDelhi - Pg. no : 589 – 590

•Keith Wilson, John Walker – Practical Biochemistry, Principles and Techniques – fifth edition – 2000 – Cambridge university Press - Pg. no : 592 – 595, 586, 602

•P.K. Gupta – Biotechnology and Genomics – first edition – 2010 – Rastogi publications – Meerut - Pg. no : 83 - 87