MALDI - TOF

DESCRIBES ON THE PRINCIPLE, WORKING, INSTRUMENTATION & APPLICATIONS OF MALDI- TOF

1. MALDI - TOF HIMA HARIDASAN

2. INTRODUCTION Proteome – complete set of proteins encoded in a genome Proteomics Protein identification requires their separation by methods :_ IEF SDS- gel electrophoresis Mass spectroscopy (MS)

3. MASS SPECTROSCOPY Separation of charged molecules/ molecules according to their mass to charge Determines relative molecular mass ( M r ) High resolution, precision, & sensitivity Requires charged gaseous molecules for its analysis

4. COMPONENTS OF MASS SPECTROSCOPY Ionization source Matrix assisted laser desorption/ ionization – time of flight( MALDI ) Electrospray (ES) Analyser m/z : TOF , Magnet,.. Detector : Photomultiplier, Electron multiplier, ..

5. COMPONENTS OF MASS SPECTROSCOPY Inlet Detector Data System Mass Analyzer High Vacuum System Ion Source Time of flight (TOF) Quadraupole Ion Trap Magnetic Sector FTMS Turbo molecular pumps HPLC Flow injection Sample plate Microchannel Plate Electron Multiplier Hybrid with photomultiplier PC Sun SPARK Station DEC Station

6. 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

7. 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 N 2 laser of 337nm or yytrium – aluminium garnet ( N d -YAG) AT 335nm. Acts as an absorbing media for the UV light

8. WORKING Sample (1 – 10 pmolmm -3 ) Mixing Excess matrix Sample – matrix Dried on target plate Co- crystallization UV rays Desorption Pulses of laser light Rapid excitation

9. 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

10. Sample molecule ionization MH + + A - > M + AH + (M - H) - + A - > [A – H - ] + M

11. MALDI –IONIZATION MECHANISM

12. 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

13. 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

14. TOF Best type of mass analyser to couple to MALDI Unlimited mass range Macromolecules of M r > 400,000 – accurately measured 100 pulses of laser light (10nanoseconds) Result – seen as a good spectrum Camera – tracks the laser beam around the MALDI

15. 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

16. Principle :- Ions Enter Flight tube Reaches Detector Acceleration of ions Fixed point & initial time Ion separation (according to m/z ratio) TOF Converted to Mass

17. 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

18. DE MALDI – TOF MS Delayed extraction TOF MS Mass dependent Used for DNA sequencing Lacks resolution & sentivity to analyze complex mixtures

19. 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

20. 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.

21. 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

22. MALDI – TOF INSTRUMENT COMPONENTS Sample target plate High vacuum chamber Camera Laser beam source Clock Flight tube Deflector Data system Reflectron

23. Camera Laser Sample plate Pumping Pumping Timed ion selector Reflector Linear detector Extraction grids Reflector detector

24. WORKING OF THE COMPONENTS Sample-matrix Target plate High vacuum chamber Camera tracks laser beam Irradiation with laser pulses

25. 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 )

26. TYPES OF MALDI SAMPLE PLATES 100 well stainless steel flat plates Four – hundred – spot Teflon – coated plates Gold - coated plates

27. 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 + I 2 + ……..I n TIC – to measure during online MS

28. REFLECTRON Reflector An ion mirror that provides higher resolution Increases overall path length for an ion Corrects minor variation in the energy spread of ions of 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.

29. 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.

30. APPLICATIONS

31. CONCLUSION MALDI – TOF Working Measures only mass Instrument components Working of the instruments

32. 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

33. 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