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THE SCIENCE BEHIND MASS SPECTROMETRY (Biology of a Chemistry Machine) SARAVANAN KUMAR , Research Associate , Proteomics Fa...
Thanks to them
Mass Spectrometry
Mass Spectrometry/meter..... Mass spectrometry – Science – based on the  motion  of  charged  particles in a    electric o...
Inside the MS meter ,Ionisation.... ESI- Electro spray ionisation LDI –Laser desorption ionisation
MALDI - acceleration....
ESI - acceleration....
Electro spray & Laser desorption
<ul><li>Laser ablation :  Process of removing material from a solid (or occasionally liquid) surface by irradiating it wit...
Physics contd….. <ul><li>The charged analyte which can respond to field is ready for flight. </li></ul><ul><li>Flight: It ...
Now Chemistry.....!! <ul><li>The analyte (Peptide,protein,oligo....) is dissolved in UV absorber (Phy-Che) </li></ul><ul><...
Some Mathematics now…. <ul><li>MALDI data: </li></ul><ul><li>No of possible peaks for 15.2 kd (15200 da) protein and their...
Mass calculation ESI -MS m/z= (M+n) /n
m/z= (MW+n) /n m/z=(MW+n+1) / (n+1) m/z= (MW+n) /n
MALDI MS <ul><li>m = 14 </li></ul><ul><li>M= 14 ?  - C+H(2) = 14 </li></ul><ul><li>Highest = 86 </li></ul><ul><li>86/14= 6...
A Short break……. <ul><li>LASER ablation in MALDI, in ESI…….  </li></ul><ul><li>MALDI-Matrix Assisted Laser Desorption Ioni...
Finally Biology.....   <ul><li>What is the information from a PMF data? </li></ul><ul><li>Protein ID- name only nothing mo...
ESI-MS of peptide
ESI MS of Protein
MALDI MS of Protein 0.0 0.2 0.4 0.6 0.8 1.0 4 x10 Intens. [a.u.] 15000 20000 25000 30000 35000 40000 45000 50000 55000 m/z...
MALDI MS of Peptide Diff m/z= 22 da 2196.128 2218.106
Tandem Mass spectrometry (MS/MS) <ul><li>Plot of back bone ions; a,b,c(N to C) and x,y,z (C toN). </li></ul><ul><li>Look f...
Peptide sequence based on Backbone ions Peptide ion formation
PTM identification/prediction
<ul><li>From Mass spectrum: </li></ul><ul><li>Relative abundance and not absolute </li></ul><ul><li>Mass spectrum does not...
For the Real time Experts (Bench workers) http://www.abrf.org/index.cfm/dm.home
 
Some tips……  <ul><li>Urea + heat + protein = carbamylation,  m 41 da </li></ul><ul><li>Concentration of protein loaded in ...
Possible solutions for some common problems…. <ul><li>Salt, detergent in the sample- employ RP principle…. HPLC?? </li></u...
LC-ESI MS
LC-MALDI -MS
Final slide or tips…. <ul><li>TPCK-Tosyl phenylalanyl chloromethyl ketone (trypsin) </li></ul><ul><li>TLCK-Tosyl-Lys-chlor...
<ul><li>Data presented: Work done by  </li></ul><ul><li>Saravanan Kumar in  </li></ul><ul><li>ICGEB for Dr.V.S.Reddy </li>...
Queries & Acknowledgements to: <ul><li>Mr.Saravanan Kumar  ,  </li></ul><ul><li>Research Associate, </li></ul><ul><li>Prot...
You need to be aware of what others are doing, applaud their efforts, acknowledge their successes, and encourage them in t...
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Saravanan Kumar,V.Siva Reddy - Workshop on Proteomics

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Transcript of "Saravanan Kumar,V.Siva Reddy - Workshop on Proteomics"

  1. 1. THE SCIENCE BEHIND MASS SPECTROMETRY (Biology of a Chemistry Machine) SARAVANAN KUMAR , Research Associate , Proteomics Facility, Plant Transformation Group, International Centre for Genetic Engineering & Biotechnology, New Delhi National Agricultural Innovation Project (NAIP) supported workshop on Proteomics: High Throughput Analysis of Proteins and Proteome by Mass Spectrometry” 22 nd - 26 th March 2010 ICGEB, New Delhi DR. V. SIVA REDDY , Organizer & Consortium Principal Investigator , NAIP component -4 , International Centre for Genetic Engineering & Biotechnology, New Delhi
  2. 2. Thanks to them
  3. 3. Mass Spectrometry
  4. 4. Mass Spectrometry/meter..... Mass spectrometry – Science – based on the motion of charged particles in a electric or magnetic field Motion- movement- mass/charge (m/z) z- if the charge is known, the mass of the ion is known m/z – m + or (m+H) + m/z= (M+n) /n M= (m/z)*n-n Transfers sample in to vacuum Ionization/ acceleration Flight/ resolution Signal detection
  5. 5. Inside the MS meter ,Ionisation.... ESI- Electro spray ionisation LDI –Laser desorption ionisation
  6. 6. MALDI - acceleration....
  7. 7. ESI - acceleration....
  8. 8. Electro spray & Laser desorption
  9. 9. <ul><li>Laser ablation : Process of removing material from a solid (or occasionally liquid) surface by irradiating it with a  laser  beam. </li></ul><ul><li>LASER is the source- the “ mechanism” – Electromagnetic radiation-UV/IR- light in the ultraviolet range </li></ul><ul><li>Plasma : Due to ablation plasma is formed. Plasma  is a  gas  in which a certain portion of the particles are  ionized . </li></ul><ul><li>Plasma has charged carriers (ions),it is electrically conductive . </li></ul>Inside the Meter..... Some Physics!! Ions can respond to electric or magnetic field
  10. 10. Physics contd….. <ul><li>The charged analyte which can respond to field is ready for flight. </li></ul><ul><li>Flight: It depends on various factors, </li></ul><ul><li>Vacuum-Mean free path, λ . </li></ul>
  11. 11. Now Chemistry.....!! <ul><li>The analyte (Peptide,protein,oligo....) is dissolved in UV absorber (Phy-Che) </li></ul><ul><li>The MATRIX: it’s a </li></ul><ul><li>UV absorber </li></ul><ul><li>Energy mediator for </li></ul><ul><li>ionisation </li></ul><ul><li> </li></ul>
  12. 12. Some Mathematics now…. <ul><li>MALDI data: </li></ul><ul><li>No of possible peaks for 15.2 kd (15200 da) protein and their m/z values ? </li></ul><ul><li>Atleast three, singly charged monomer (M+H) - 15201 da </li></ul><ul><li> doubly charged monomer(M+2H) - 7601 da </li></ul><ul><li> singly charged dimer (2M+H) - 30401 da </li></ul><ul><li>For peptides: Singly charged </li></ul><ul><li>3-5 isotopic peaks </li></ul><ul><li>Sodium adducts (M+Na)+ 22 da </li></ul><ul><li>Potassium adducts (M+K) + 38 da </li></ul><ul><li>ESI data : </li></ul><ul><li>(M+ 56H)56+ = 840.3 m/z </li></ul><ul><li>What is the mass of this molecule? </li></ul><ul><li>(840.3 * 56)- 56 = 47000.8 da </li></ul>m/z= (M+n) /n
  13. 13. Mass calculation ESI -MS m/z= (M+n) /n
  14. 14. m/z= (MW+n) /n m/z=(MW+n+1) / (n+1) m/z= (MW+n) /n
  15. 15. MALDI MS <ul><li>m = 14 </li></ul><ul><li>M= 14 ? - C+H(2) = 14 </li></ul><ul><li>Highest = 86 </li></ul><ul><li>86/14= 6.142... </li></ul><ul><li>There may be 6 C </li></ul><ul><li>6* 12 =72 </li></ul><ul><li>86-72= 14 </li></ul><ul><li>C 6 H 14 = Hexane </li></ul><ul><li>Its simple..... </li></ul>
  16. 16. A Short break……. <ul><li>LASER ablation in MALDI, in ESI……. </li></ul><ul><li>MALDI-Matrix Assisted Laser Desorption Ionisation, ESI…… </li></ul><ul><li>N 2 nebulization in ESI, aiding droplet formation and desolvation </li></ul><ul><li>Pneumatically Assisted Electrospray Ionization </li></ul>
  17. 17. Finally Biology..... <ul><li>What is the information from a PMF data? </li></ul><ul><li>Protein ID- name only nothing more….. </li></ul><ul><li>Species, Molecular weight, Isoelectric point etc are database information and not the information from the sample….. Getting it. </li></ul><ul><li>Protein identification (PMF): Correlation of the information obtained from MS and the information contained in protein/DNA sequence databases. </li></ul><ul><li>Parameters to be noted: </li></ul><ul><li>error tolerance <150ppm (0.3da) (MALDI data) </li></ul><ul><li> no of peptides/ sequence coverage </li></ul><ul><li>Intensity coverage. </li></ul>
  18. 18. ESI-MS of peptide
  19. 19. ESI MS of Protein
  20. 20. MALDI MS of Protein 0.0 0.2 0.4 0.6 0.8 1.0 4 x10 Intens. [a.u.] 15000 20000 25000 30000 35000 40000 45000 50000 55000 m/z 26661.395 53321.02 13331.20
  21. 21. MALDI MS of Peptide Diff m/z= 22 da 2196.128 2218.106
  22. 22. Tandem Mass spectrometry (MS/MS) <ul><li>Plot of back bone ions; a,b,c(N to C) and x,y,z (C toN). </li></ul><ul><li>Look for the no of back bone ions identifed. </li></ul><ul><li>Modified ions, eg:Phosphorylation- mass of an </li></ul><ul><li>back bone ion from an amino acid + 80 da </li></ul><ul><li>No of identified ions vs no of predicted ions (theoretical). </li></ul><ul><li>FDR ?, significance. </li></ul><ul><li>Peptide fragment mass tolerance:<1 da. </li></ul><ul><li>Restriction in protein molecular weight etc. </li></ul>
  23. 23. Peptide sequence based on Backbone ions Peptide ion formation
  24. 24. PTM identification/prediction
  25. 25. <ul><li>From Mass spectrum: </li></ul><ul><li>Relative abundance and not absolute </li></ul><ul><li>Mass spectrum does not give a quantitative information. </li></ul><ul><li>Quantitative: Labelling chemistry - ICPL,ITRAQ etc. </li></ul><ul><li>Protein ID depends on protein conc. </li></ul><ul><li>Sequence coverage depends on biochem of protein… </li></ul>A Kind Reminder for Biologist by Mass spectrometrist......
  26. 26. For the Real time Experts (Bench workers) http://www.abrf.org/index.cfm/dm.home
  27. 28. Some tips…… <ul><li>Urea + heat + protein = carbamylation, m 41 da </li></ul><ul><li>Concentration of protein loaded in gel for identification, sequencing etc </li></ul><ul><li>Temperature of the sealing agarose in case of 2D !!?? </li></ul><ul><li>Staining – does the stain modify the protein, if so what is the modification, what could be the change in mass, </li></ul><ul><li>is it global or variable. </li></ul><ul><li>Stability of trypsin or any protease in the digestion mixture </li></ul><ul><li>Grade of the protease used, Proteomics grade?.... </li></ul><ul><li>% of ACN and TFA used for peptide extraction after ingel digestion </li></ul><ul><li>Nature of the peptide pellet after concentration, peptide pellets are transparent and not dense (opaque). </li></ul>
  28. 29. Possible solutions for some common problems…. <ul><li>Salt, detergent in the sample- employ RP principle…. HPLC?? </li></ul><ul><li>Autolysis of protease, poor PMF pattern. </li></ul><ul><li>stabilize the protease by adding ions Ca for Trypsin, check the pH, temp cond. </li></ul><ul><li>Stain retained in peptide pellet… it happens. </li></ul><ul><li>Pass it through a Ziptip etc, or consult a expert… </li></ul>Zip tips, spin columns, microcons
  29. 30. LC-ESI MS
  30. 31. LC-MALDI -MS
  31. 32. Final slide or tips…. <ul><li>TPCK-Tosyl phenylalanyl chloromethyl ketone (trypsin) </li></ul><ul><li>TLCK-Tosyl-Lys-chloromethylketone (chymotrypsin) </li></ul><ul><li>Oxidation of Methionine (to Sulphoxide) - 16 da </li></ul><ul><li>Oxidation of Methionine (to Sulphone) - 32 da </li></ul><ul><li>Carbamylation - 41 da </li></ul><ul><li>Carboxyamidomethyl - 160 da </li></ul><ul><li>Carbamidomethyl - 57 da </li></ul><ul><li>Sodium - 22 da </li></ul><ul><li>Potassium - 38 da common modification in synthetic peptides </li></ul><ul><li>100 ppm - 0.2 da </li></ul><ul><li>MALDI TOF/TOF error tolerance - 200 ppm ( rarely- peptide)(<100 ppm) - 0.75 da (rarely- peptide fragments) </li></ul><ul><li>Coomassie - 833.048 da </li></ul><ul><li>Mass spec is very simple machine, need not to worry MUCH….. </li></ul>
  32. 33. <ul><li>Data presented: Work done by </li></ul><ul><li>Saravanan Kumar in </li></ul><ul><li>ICGEB for Dr.V.S.Reddy </li></ul><ul><li>IGIB,TCGA,JNU for Dr.H.R.Das </li></ul>
  33. 34. Queries & Acknowledgements to: <ul><li>Mr.Saravanan Kumar , </li></ul><ul><li>Research Associate, </li></ul><ul><li>Proteomics Facility, </li></ul><ul><li>Plant Transformation Group, </li></ul><ul><li>International Centre for Genetic Engineering & Biotechnology, </li></ul><ul><li>New Delhi-110067 </li></ul><ul><li>[email_address] </li></ul><ul><li>Dr.V.Siva Reddy , </li></ul><ul><li>Group Leader, </li></ul><ul><li>Plant Transformation Group, </li></ul><ul><li>International Centre for Genetic Engineering & Biotechnology, </li></ul><ul><li>New Delhi-110067 </li></ul><ul><li>[email_address] </li></ul>
  34. 35. You need to be aware of what others are doing, applaud their efforts, acknowledge their successes, and encourage them in their pursuits. When we all help one another, everybody wins.

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