workshop sk-vsr 2010

8/7/2019 workshop SK-VSR 2010

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National Agricultural Innovation Project (NAIP) supported workshop on

Proteomics: High Throughput Analysis of Proteins and Proteomeby Mass Spectrometry

22nd - 26th March 2010

ICGEB, New Delhi

(Biology of a Chemistry Machine)

SARAVANAN KUMAR,

Research Associate,

Proteomics Facility, Plant Transformation Group,International Centre for Genetic Engineering &

Biotechnology, New Delhi

DR. V. SIVA REDDY,

Organizer & Consortium Principal Investigator,

NAIP component -4 ,International Centre for Genetic Engineering &

Biotechnology, New Delhi


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Thanks to them


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Mass Spectrometry


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Inside the MS meter ,Ionisation....

ESI- Electro spray ionisation LDI Laser desorption ionisation


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MALDI - acceleration....


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ESI - acceleration....


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Electro spray & Laser desorption


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Laser ablation : Process of removing material from a solid (oroccasionally liquid) surface by irradiating it with a laser beam.

LASER is the source- the mechanism Electromagneticradiation-UV/IR-light in the ultraviolet range

Inside the Meter..... Some Physics!!

Plasma: Due to ablation plasma is formed. Plasma is a gas in

which a certain portion of the particles are ionized. Plasma has charged carriers (ions),it is electrically conductive.

Ions can respond to electric or magnetic field


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Physics contd..

The charged analyte which can respond to

field is ready for flight.

Flight: It depends on various factors,

Vacuum-Mean free path,.


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Now Chemistry.....!! The analyte (Peptide,protein,oligo....) is dissolved in UV

absorber (Phy-Che)

The MATRIX: its aUV absorber

Energy mediator for


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Some Mathematics now. MALDI data: No of possible peaks for 15.2 kd (15200 da) protein and their

m/z values ?

Atleast three, singly charged monomer (M+H) - 15201 dadoubly charged monomer(M+2H) - 7601 da

singly charged dimer (2M+H) - 30401 da

For peptides: Singly charged

3-5 isotopic peaksSodium adducts (M+Na)+ 22 da

Potassium adducts (M+K) + 38 da

ESI data :

(M+ 56H)56+ = 840.3 m/z

What is the mass of this molecule?

(840.3 * 56)- 56 = 47000.8 da

m/z= (M+n) /n


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Mass calculationESI -MS

m/z= (M+n) /n


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m/z= (MW+n) /n

m/z=(MW+n+1) / (n+1)

m/z= (MW+n) /n


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A Short break.

LASER ablation in MALDI, in ESI.

MALDI-Matrix Assisted Laser Desorption Ionisation,ESI

N2 nebulization in ESI, aiding droplet formation and

desolvation

Pneumatically Assisted Electrospray Ionization


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Slide 16

SK-VSR1 , 2/3/2011


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Finally Biology.....

Protein identification (PMF): Correlation of the informationobtained from MS and the information contained inprotein/DNA sequence databases.

Parameters to be noted:error tolerance


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ESI-MS of peptide


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ESI MS of Protein


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0.6

0.8

1.0

4x10

Intens.

[a.u.

]

26661.395

MALDI MS of Protein

0.0

0.2

0.4

15000 20000 25000 30000 35000 40000 45000 50000 55000m/z

53321.02

13331.20


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2196.128

2218.106

2

3

4x10

I

ntens.

[a.u.]

MALDI MS of Peptide

Diff m/z= 22 da

2196.128 2218.106

2202.361

2210.086

0

1

2185 2190 2195 2200 2205 2210 2215 2220m/z


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Tandem Mass spectrometry (MS/MS)

Plot of back bone ions; a,b,c(N to C) and x,y,z (C toN).

Look for the no of back bone ions identifed.

Modified ions, eg:Phosphorylation- mass of anback bone ion from an amino acid + 80 da

No of identified ions vs no of predicted ions

(theoretical).

FDR ?, significance.

Peptide fragment mass tolerance:


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Peptide sequence based on Backbone ions

Peptide ion formation


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PTM identification/prediction


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A Kind Reminder for Biologist

by Mass spectrometrist...... From Mass spectrum:

Relative abundance and not absolute

Mass spectrum does not give a quantitativeinformation.

Quantitative: Labelling chemistry- ICPL,ITRAQetc.

Protein ID depends on protein conc.

Sequence coverage depends on biochem ofprotein


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For the Real time Experts (Bench workers)http://www.abrf.org/index.cfm/dm.home


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Some tips

Urea + heat + protein = carbamylation, m 41 da Concentration of protein loaded in gel for identification,

sequencing etc

Temperature of the sealing agarose in case of 2D !!??

Staining does the stain modify the protein, if so what is

the modification, what could be the change in mass,

.

Stability of trypsin or any protease in the digestion mixture

Grade of the protease used, Proteomics grade?....

% of ACN and TFA used for peptide extraction after ingel

digestion Nature of the peptide pellet after concentration, peptide

pellets are transparent and not dense (opaque).


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Possible solutions for some common

problems. Salt, detergent in the sample- employ RP

principle. HPLC??

Autolysis of protease, poor PMF pattern.stabilize the protease by adding ions Ca for

Zip tips, spin columns, microcons

, , .

Stain retained in peptide pellet it happens.

Pass it through a Ziptip etc, or consult a expert


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LC-ESI MS


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LC-MALDI -MS

Fi l lid i


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Final slide or tips.

TPCK-Tosyl phenylalanyl chloromethyl ketone (trypsin)

TLCK-Tosyl-Lys-chloromethylketone (chymotrypsin)

Oxidation of Methionine (to Sulphoxide) - 16 da

Oxidation of Methionine (to Sulphone) - 32 da

Carbamylation - 41 da

Carboxyamidomethyl - 160 da Carbamidomethyl - 57 da

Sodium - 22 da

Potassium - 38 da common modification in synthetice tides

100 ppm - 0.2 da

MALDI TOF/TOF error tolerance - 200 ppm ( rarely- peptide)(


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Data presented: Work done by

Saravanan Kumar in

ICGEB for Dr.V.S.Redd

IGIB,TCGA,JNU for Dr.H.R.Das

Queries & Acknowledgements to:


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Queries & Acknowledgements to: Dr.V.Siva Reddy,

Group Leader,

Plant Transformation Group,

International Centre for Genetic Engineering & Biotechnology,

New [email protected]

Mr.Saravanan Kumar ,Research Associate,

Proteomics Facility,

Plant Transformation Group,International Centre for Genetic Engineering

& Biotechnology,

New Delhi-110067

[email protected]


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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.

workshop sk-vsr 2010

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