(I) Basics of MALDI-TOF (II) Sample preparation (III ... · 12/11/12 1 Dr.$Sanjeeva$Srivastava$ IIT$Bombay$ IIT Bombay Proteomics Course NPTEL • (I) Basics of MALDI-TOF • (II)

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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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(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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Sam

ple

Pla

te

Analyte Laser Beam

Matrix Extraction Grid

TOF


•  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

IIT Bombay Proteomics Course NPTEL 7 7

•  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



An overview of MALDI-TOF analysis: Animation

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(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


•  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


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


•  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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Sample clean-up using ZipTip: Video


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


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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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


•  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


•  (I) Basics of MALDI-TOF

•  (II) Sample preparation

•  (III) MALDI instrumentation

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•  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)


•  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

Documents

(I) Basics of MALDI-TOF (II) Sample preparation (III ... · 12/11/12 1 Dr.$Sanjeeva$Srivastava$ IIT$Bombay$ IIT Bombay Proteomics Course NPTEL • (I) Basics of MALDI-TOF • (II)