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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,
Mr.Saravanan Kumar ,Research Associate,
Proteomics Facility,
Plant Transformation Group,International Centre for Genetic Engineering
& Biotechnology,
New Delhi-110067
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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.