• Gel-based proteomics • 2-DE work-flow • New methods for proteomics applications

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Dr. Sanjeeva Srivastava IIT Bombay

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•  Gel-based proteomics

•  2-DE work-flow

•  New methods for proteomics applications

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IIT Bombay Proteomics Course NPTEL

•  Challenges of 2-DE

•  2-D Fluorescence Difference Gel Electrophoresis (DIGE)

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pI

MW

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Representative 2-DE gel – Bacterial (B. subtilis) proteome

pI

MW

5

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•  Gel artifacts and gel-to-gel variation

•  samples, electrophoresis, staining

•  Image analysis

•  defining spot boundaries

•  user bias in data analysis

•  Biological and technical variations

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TCA-acetone precipitation,

TCA in pellet

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Plant protein extraction, solubilization without precipitation

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Crude sample

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Carbamylation train Impurities in urea

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Old TEMED

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Tris impurity

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Salts

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Vertical streaking (pH 4-7)

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Overloading with highly abundant proteins in serum

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Only equilibration with DTT

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20 min DTT / 30 min IAA

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Under-focusing

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Over-focusing

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Inherent Induced

Between individuals

Between biological

states

Eﬀects which can’t be controlled

Eﬀects which can be controlled (e.g. sample

prep) Biological

Technical

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•  To overcome intrinsic gel-to-gel variability of 2-DE, the DIGE multiplexing technology emerged

•  This method was reported by Unlu et al. 1997

•  Electrophoresis 1997, 18, 2071–2077

•  A protein labelling and separation technique

•  Optical detection of proteins with a fluorescent tag

•  Linear detection in wide range of protein abundance

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•  Commonly used fluorescent protein labelling reagents for DIGE

•  N-hydroxysuccinimidyl (NHS) ester derivatives of cyanine dyes Cy2, Cy3 and Cy5

•  Size and charge matched - labeled samples co-migrate within gel

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IIT Bombay

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Proteomics Course NPTEL

NHS-Cy3 NHS-Cy5 NHS-Cy2

MW - 582.8 Da Excitation – 540 nm

Emission – 590 nm

MW - 580.7 Da Excitation – 620 nm

Emission – 680 nm

MW - 550.6 Da Excitation – 480 nm Emission – 530 nm

•  Photostable

•  pH insensitive

•  Spectrally distinct

•  Minimum labeling

•  Lysine labeling

•  Charge and size matched, no multiple labels

•  labelling of 3% of all proteins, Cy2, Cy3, Cy5

•  Saturation labeling

•  Cysteine labeling

•  Charge neutral and size matched, multiple labels

•  Very sensitive, only Cy3 and Cy5

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%

Distribution of amino acids across the proteins

IIT Bombay

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Lysine is preferred amino acid to label, it’s frequency is >5%;

statistically almost every protein has at least one lysine residue

•  Cyanine dyes react with primary amine groups of target proteins

•  N-terminal α-amino and lysine ε-amino groups

•  Nucleophilic substitution

•  All available lysine labelling would create very hydrophobic proteins

•  Minimal labelling - only 3% of whole proteome is

labeled

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•  Structure of CyDye Fluor comprises

•  positive charged CyDye fluorophore, linker, NHS ester reactive group

•  At pH 8.5 NHS ester couples with amine group of lysine in proteins, forms covalent amide bond

IIT Bombay

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•  The amine group of a lysine residue is positively charged at neutral or acidic pH’s.

•  Dye is also positively charged, so the net charge pI is effectively unchanged

•  Cy3 and Cy5 are two spectrally resolvable dyes

matched for mass and charge

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•  CyDyes - reconstitute in Dimethyformamide (DMF)

•  Lysis buffer:

•  30 mM Tris, 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, pH 8.5

•  No IPG buffers or carrier ampholytes; no reductant

•  50 µ g protein label with 400 pmoles of dye on ice- water for 30 min

•  Reactions are quenched with excess of primary amine (free lysine - 1 µL of 10 mM lysine)

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1 2 3 4 1 2 3 4

Controls Treatment

Pooled internal control

•  Pooled internal standard preparation by combining aliquot of equal amounts of protein from each samples in experiment

IIT Bombay

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•  Every protein in population appears on each gel

•  Gel-to-gel variation reduces

•  Gel-to-gel match and comparison easier

•  A reference point for every protein species on each gel in the experiment

•  Automated accurate spot statistics can be

performed

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Internal standard

Sample 1 Sample 2

Micropipette

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IIT Bombay

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Cy3 Cy5 Cy2

Internal standard

Sample 1 Sample 2

Micropipette

Internal

standard

Sample 1 Sample 2

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IIT Bombay

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pH 3

pH 9

Tracking dye

Direction of

migration

Isoelectric focussing

unit

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IIT Bombay

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The IPG strip is then placed on the SDS-polyacrylamide gel for separation in the second dimension.

SDS-PAGE slab 1

^st

Equilibration –

Gel placed in DTT

2

^nd

Equilibration- Gel placed in iodoacetamide

Direction of migration,

Gel scanning

Cy3 Labeled Proteins

Cy5 Labeled Proteins Cy2

Labeled

Proteins

DIGE Imager

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IIT Bombay

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Cy3

Cy2

Cy5 Sample 1

Standard (1+2)

Sample 2

Mix

Cy3 Excitation

Cy5 Excitation

Cy2 Excitation

Pro te in Sa mp le s

Cyanine dyes

Cy3 Cy5 Cy2

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Overlapped Combined

IIT Bombay

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3 images from 1 gel

DIA - Co-detection of 3 images

BVA - Multiple gel matching

IIT Bombay

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1 2

D is eas e 3 H eal th y P ath oge n 2 P ath oge n 1

Spot 1 Spot 2

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Gel 03 Gel 06

Gel 01

Gel 02

Gel 04

Gel 05

HC Pf HC Pf

IIT Bombay

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•  Principal component - a linear combination of optimally-weighted observed variables

•  PCA analysis to test, whether:

•  Protein expression is uniform in multiple sample from same experimental group

•  If results are consistent, spot map from same

experimental group located in same area

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IIT Bombay

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•  Discriminant analysis - To classify unknown data to

known classes

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Items (proteins, spot maps, experimental groups) are grouped together in an hierarchical way

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•  Which of these two would be the better technique to separate serum protein samples obtained from 250 patients in a clinical trial?

2-D gel

electrophoresis (2-DE) Difference gel

electrophoresis (DIGE)

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IIT Bombay

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•  Gel to gel variation is overcome by separating proteins on single gel

•  Large sample variations can be reduced by internal standard

•  User bias in data analysis can be eliminated by co-detection method

•  Challenges associated with 2-DE

•  Fluorescence Difference Gel Electrophoresis

•  Labeling, Sample preparation, step-by-step procedure

•  Comparison of 2-DE vs. DIGE

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•  Unlü M., Morgan EM, Minden JS: Difference gel electrophoresis: a single gel method for detecting changes in protein extracts.

Electrophoresis 1997, 18, 2071-2077

•  Van den Bergh, G., Arckens, L.: Fluorescent 2-D DIGE unveils the potential of gel-based proteomics. Current Opinion in Biotechnology, 2004, 15, 1-6

•  Patton, W.F.: Detection technologies in proteome analysis. J Chromatogr B, 2002, 771, 3- 31

•  Yuguang Wang, Haiying Li, Sixue Chen. Advances in quantitative proteomics. Frontiers in Biology. June 2010, Volume 5, Issue 3, pp 195-203.

Dr. Srinivas and Lalith Kishore, GE healthcare for

sharing information related to 2D-DIGE

• Gel-based proteomics • 2-DE work-flow • New methods for proteomics applications

Dr. Sanjeeva Srivastava IIT Bombay

• Gel artifacts and gel-to-gel variation

Crude sample

Overloading with highly abundant proteins in serum

Eﬀects which can be controlled (e.g. sample

• Commonly used fluorescent protein labelling reagents for DIGE

• Charge and size matched, no multiple labels

statistically almost every protein has at least one lysine residue

• Structure of CyDye Fluor comprises

• Dye is also positively charged, so the net charge pI is effectively unchanged

• Reactions are quenched with excess of primary amine (free lysine - 1 µL of 10 mM lysine)

• A reference point for every protein species on each gel in the experiment

SDS-PAGE slab 1

Direction of migration,

Gel scanning

D is eas e 3 H eal th y P ath oge n 2 P ath oge n 1

Gel 03 Gel 06

• Principal component - a linear combination of optimally-weighted observed variables

known classes

electrophoresis (DIGE)

• Comparison of 2-DE vs. DIGE

• Patton, W.F.: Detection technologies in proteome analysis. J Chromatogr B, 2002, 771, 3- 31

•  Gel artifacts and gel-to-gel variation

•  Commonly used fluorescent protein labelling reagents for DIGE

•  Charge and size matched, no multiple labels

•  Structure of CyDye Fluor comprises

•  Dye is also positively charged, so the net charge pI is effectively unchanged

•  Reactions are quenched with excess of primary amine (free lysine - 1 µL of 10 mM lysine)

•  A reference point for every protein species on each gel in the experiment

•  Principal component - a linear combination of optimally-weighted observed variables

•  Comparison of 2-DE vs. DIGE

•  Patton, W.F.: Detection technologies in proteome analysis. J Chromatogr B, 2002, 771, 3- 31