(i) basics of maldi-tof (ii) sample preparation (iii ... · • 50 mm ammonium bicarbonate (50µl)...
TRANSCRIPT
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Dr. Sanjeeva Srivastava IIT Bombay
IIT Bombay Proteomics Course NPTEL
• (I) Basics of MALDI-TOF
• (II) Sample preparation • In-gel digestion
• Zip-tip sample clean-up
• Matrix and sample plating
• (III) MALDI instrumentation
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IIT Bombay Proteomics Course NPTEL
(I) Basics of MALDI-TOF
IIT Bombay Proteomics Course NPTEL 4
Sam
ple
Pla
te
Analyte Laser Beam
Matrix Extraction Grid
TOF
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IIT Bombay Proteomics Course NPTEL 5
Sam
ple
Pla
te
Analyte Laser Beam
Matrix Extraction Grid
TOF
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• Merits
• Sample preparation easy
• More tolerant to salts than ESI
• Produces mainly singly charged ions
• Demerits
• Strong dependence on sample preparation methods
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Reflector
Detector
Source
LASER Ion Source Ionisation Sample
Ion Current
TOF
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• TOF mass analyzer consists of ion acceleration and focusing optics and a flight tube
• Measures m/z ratios of ions based on time it takes for ions to fly in analyzer & strike the detector
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30 Kv Ion source
Laser
Sample mixed with matrix
Detector
Peptide mass > >
Time
Inte
nsity
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IIT Bombay Proteomics Course NPTEL
An overview of MALDI-TOF analysis: Animation
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IIT Bombay Proteomics Course NPTEL
(II) Sample preparation: In-gel digestion
IIT Bombay Proteomics Course NPTEL 12 Trypsin digestion
Spot excision
Reflector
Detector
LASER
TOF 1 TOF 2
Collision Cell
He He
He He
He He
He He
Pro
tein
of i
nter
est
2D –SDS-PAGE Gel
MALDI-TOF-TOF MS
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• 50 mM ammonium bicarbonate (50µL) & 50µL ACN • incubate (37°C, 10 min) and aspirate the solution
• dispense 50µL of ACN and incubate (37°C, 5 min) • aspirate the solution and re-incubate (37°C, 10 min)
• dispense 50µL of 10 mM DTT • incubate (37°C, 20 min)
Coomassie destain
Dehydration
Reduction
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• dispense 30µL of 55 mM iodoacetamide • incubate at (37°C, 20 min) and aspirate the solution
• dispense 50µL of acetonitrile • incubate (37°C, 5 min) and aspirate the solution • remove residual ACN by incubation (37°C, 5 min)
• dispense 15µL of trypsin solution • incubate (RT, 10 min) to allow trypsin to absorb into gel
• add 15 µL of 50 mM ammonium bicarbonate, incubate (37°C, 4 h)
Alkylation
Dehydration
Digestion
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NH3 COO- S
O
S
CH2CNH2
O
IAA, 30°C
DTT, 65°C
NH3 COO-
S S
NH3 COO- S
H
S
H
CH2CNH2
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Trypsin digest
Lysine
Arginine or (K/R) (K/R) P C N
1 2 3 4
N (K/R) 1
(K/R) 2
Cleaves on the C-terminal of K or R residues
(K/R) P C 3 4
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In-gel digestion: Video
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• In-gel digested protein samples are processed further using ZipTip pipette tips containing C18 or C4 media for enrichment of peptides
• Salts and interfering agents, detergents are washed and finally samples are eluted in a very small volume of solvent
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IIT Bombay Proteomics Course NPTEL
Sample clean-up using ZipTip: Video
IIT Bombay Proteomics Course NPTEL
Matrix selection
Matrix preparation
Sample purification
Sample deposition
Matrix deposition
Drying target
Analysis
Sample & Matrix Preparation
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Peptides less than 5000 daltons, lipids and nucleic acids
Peptides and proteins having higher than 5000 daltons and sometimes also use for lipids
OH
OH C
N
O
α-cyano-4-hydroxycinnamic acid (α-cyano)
OH
OH
O
O
O
Sinapinic acid
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Small molecules and peptides which are not ionized by other matrices
Used for small nucleotides and phosphorylation studies on proteins
Generally used for nucleotides
OH H O
OH
O
2,5-Dihydroxybenzoic acid (DHB)
OH
OH OH
O
Trihydroxyacetophenone (THAP) O
OH
Picolinic acid
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IIT Bombay Proteomics Course NPTEL
Matrix preparation is done by mixing matrix into a suitable solvent and vortex for few minutes to dissolve it properly
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(III) MALDI-TOF instrumentation: Video
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Reflector
Detector
Source
LASER Ion Source Ionisation
Ion Current
TOF 1 TOF 2
Collision Cell
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• MALDI can be coupled to tandem TOF-TOF or hybrid Q-TOF analyzers, separated by a collision cell
• Much higher sensitivity than TQ and single TOF
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• (I) Basics of MALDI-TOF
• (II) Sample preparation
• (III) MALDI instrumentation
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IIT Bombay Proteomics Course NPTEL
• Lee, T.A. A beginner’s guide to mass spectral interpretation. John Wiley & sons Ltd. 1998. ISBN: 0 471 97629 6.
• Stroobant. V. and de Hoffmann. E. Mass spectrometry: principles and applications. 2007. John Wiley & sons Ltd. ISBN 978-0-470-03311-1.
• Ekman. R., Silberring. J., Westman-Brinkmalm. A. and Kraj. A. Mass spectrometry: Instrumentation, Interpretation, and Applications. 2009. John Wiley & sons Ltd. ISBN 978-0-471-71395-1.
• Andrej Shevchenko, Henrik Tomas, Jan Havli, Jesper V Olsen & Matthias Mann. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature Protocols 1, 2856 - 2860 (2007)
IIT Bombay Proteomics Course NPTEL
• Medzihradszky KF, Campbell JM, Baldwin MA, Falick AM, Juhasz P, Vestal ML, Burlingame AL. The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer. Anal Chem. 2000, 72, 552-8.
• Hortin GL. The MALDI-TOF mass spectrometric view of the plasma proteome and peptidome. Clin Chem. 2006, 52, 1223-37.
• Song-Hee Im, Sung-Seen Choi. 2010. apid Communications in Mass Spectrometry. Volume 24, Issue 18, pages 2753–2758. DOI: 10.1002/rcm.4701.
• Renata A. Culak, Min Fang, Shurene Bishop Simon, Itaru Dekio, Lakshani K. Rajakaruna and Haroun N. Shah* Changes in the Matrix Markedly Enhance the Resolution and Accurate Identification of Human Pathogens by MALDI-TOF MS. J Anal Bioanal Techniques 2012, S2