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
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