An overview of 2DE technique Applications - nptel.ac.in · Plasmodium malariae Quartan Malaria IIT...

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Dr. Sanjeeva Srivastava IIT Bombay

IIT Bombay Proteomics Course NPTEL

•  An overview of 2DE technique

•  Applications •  Case study – 1

•  Case study – 2

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IIT Bombay Proteomics Course NPTEL 3

SDS PAGE

2DE DIGE

GEL ANALYSIS


Isoelectric focusing

IPG strip

Increasing pI

2DE-GEL

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Gel electrophoresis unit

SDS-Polyacrylamide Gel

Second Dimension: SDS-PAGE

Decreasing Molecular

Weight

Molecular weight


Mol

ecul

ar w

eigh

t

Spot analysis: MW and pI of protein

pH 4 pH 7 Increasing pI

Decreasing m

olecular weight

A6: Staining

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Gel1 -‐ Control Gel2 -‐ Treatment


Ray et al. Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response. J Proteomics. 2011. PMID: 22086083

Case study-1

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•  Malaria - an epidemic in 103 countries around the globe

•  Incidence of malaria worldwide ~300-500 million/ per year and death between 1.1-2.7 million people each year

•  P. vivax & P. falciparum account for 95% of malaria worldwide

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ASIA – challenge of drug resistant

strains

AFRICA – single largest cause of death

Human being Infecte

d

Liver

Infected Mosquito taking a blood meal

Sporozoites

injected

Liver Cell Infected Liver

Cell

Sporozoites

Exo-‐erythrocytic cycle in Human Liver

Ruptured Schizont

Schizont

Merozoites

Human Blood Stages

RBC

Infec9on

of RBCs

Immature tropophozoite (ring stage)

Mature Trophozite

Female Gametocyte

Male Gametocyte

Schizont

Ɨ

Ruptured Schizont

Infects normal RBCs

Healthy

Human

Normal

RBCs

Female Anopheles

Mosquito taking a blood meal

Releases Sporozoites

Gametes

Mosquito Gut

Zygote

Oocyst

Gametes

Mosquito

Salivary Gland

Ingests gametocyte

s Ruptured Oocyst

Ookinete

Macrogametocyte

Extraflagellate microgametocyte

Microgamete entering

macrogamete

Sporogenic cycle

Erythrocytic cycle

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•  P. knowlesi can also cause acute, severe illness but mortality rates are low

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Species Type of malaria

Plasmodium vivax Benign Tertian Malaria

Plasmodium falciparum Malignant Tertian Malaria

Plasmodium ovale Ovale Tertian Malaria

Plasmodium malariae Quartan Malaria


Protein extraction

Gel-based proteomics

Data analysis Protein identification

(database search)

MS analysis

Serum samples

Vivax malaria Healthy control

Validation and

characterization

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•  Patient information •  Age, Sex, Physiological status, Alcoholic patients •  Previous history of diseases •  Treatment [ If already treated; treatment information]

•  Selection of healthy control •  Pooled versus individual sample •  Process of sample collection •  Sample storage •  Reproducibility

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Blood sample

Serum separation

tube

Incubation on ice

Centrifugation Serum Storage at -80oC

Collection in small aliquots

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5 ml blood collected into butter fly syringe (kept on ice until the isolation of serum)

Centrifuged at 2500 rpm at 20°C, 10 min

Serum was collected immediately

Collected serum was divided into aliquots and stored at -20°C

Allowed to clot for 1 h


Dilution in buffer

Proteomic analysis

Classical approach

Alternative approach

High abundance protein removal

Affinity column

Ultra filtration

Desalting

High abundance serum proteins

Albumin

IgG

IgA

Hapto globin

Antitrypsin Transferrin

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Serum sample

Crude serum Desal@ng

Sonica@on &

desal@ng

Abundant protein removal, desal@ng

Serum sample IPG strip

Staining

Software analysis Protein visualization

1st dimension [IEF] 2nd dimension [SDS-PAGE]


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Crude

Sonicated-desalted

Desalted

Depleted

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4 7

CRUDE SERUM

DESALTED SERUM

SONICATED-‐DESALTED SERUM

4 7

4 7

IIT Bombay Proteomics Course NPTEL 22 Sonicated-‐desalted_silver

Desalted serum

Sonicated-‐desalted serum Coomassie

4 7

4 7

4 7

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40 µL serum was precipitated -4 volumes of ice-cold acetone containing 10% w/v TCA

incubated at -200C for 90 min

Centrifuged at 15 000 X g, 40C, for 20 min.

1 mL of ice-cold acetone was added to wash the precipitate

incubated on ice for 15 min and centrifuged as above

Acetone-containing supernatant was removed

[Urea 8M, CHAPS; 4% ; 2% IPG buffer; DTT 40mM; 1 % BPB] Pellet dissolved in lysis buffer


0

50

100

150

200

250

A B C D

Number of spots [n=3]

Cru

de

Des

alte

d

Des

alte

d an

d so

nica

ted

Des

alte

d, s

onic

ated

and

dep

lete

d

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Serum sample

Direct crude serum

TCA-‐Acetone precipita@on

Trizol extrac@on method

Sonicated desalted serum

Abundant protein removal, TCA-‐

Acetone precipita@on

Scambi et al., PLoS One 2010

Chen et al., Electrophoresis

2005

Modified from Scambi et al.,

PLoS One 2010

Lee et al., Journal of Microbiological Methods 2008

Modified from Chen et al., 2005


24 cm

66-

4 7

-400

100

600

Acetone TCA-Acetone

Spot

nu

mbe

r

66-

24 cm 4 7

[n=5]

Acetone TCA-Acetone

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4 7 24 cm 24 cm 4 7

97-

66- 55-

30-

14-

97-

66- 55-

30-

14-

Spot number Crude serum Depleted serum

Software analysis 533 719

Crude serum Depleted serum


4 7 24 cm 24 cm

Stain: Coomassie Blue

4 7

Stain: Silver

97-

66- 55-

30-

14-

97-

66- 55-

30-

14-

Spot number CBB Silver

Software analysis 719 995

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Crude Sonicated-‐desalted TCA-‐Acetone Trizol

Parameters Crude Sonicated-‐desalted

TCA-‐Acetone Trizol

1. Sonica9on -‐ + + +

2. Desal9ng -‐ + + -‐

3. Rehydra9on Ac9ve Ac9ve Passive Ac9ve

4. Amount of protein loaded

1200 µg 1200 µg 600 µg 1200 µg

5. Strip 24 cm 24 cm 24 cm 24 cm

6. Staining Soln Coomassie Coomassie Coomassie Coomassie

7. Spot Number [SoPware detected]

513 503 509 359

8. Spot Number [APer refinement]

351 363 308 208

4 7 4 7 4 7 4 7

24 cm 24 cm 24 cm 24 cm


Healthy control samples

Vivax malaria samples

Pv 1 Pv2 Pv 3 Pv4 Pv5 Pv6 Pv7 Pv8 HC 1 HC2 HC3 HC4 HC5 HC6 HC7 HC8

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4 7

24 cm 600 µg protein

BSA [66KDa]

Software detected: 539 After refinement: 392 ±10


4 7

Healthy control

4 7

P. vivax

66-

55-

30-

14-

pI

MW

pI

MW

66-

55-

30-

14-

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Soft

war

e de

tect

ed:

652

Aft

er r

efin

emen

t:

441±

10

Soft

war

e de

tect

ed:

592

Aft

er r

efin

emen

t:

372±

10

SoPware de

tected

: 592

APer re

finem

ent: 411±10

Soft

war

e de

tect

ed:

582

Aft

er r

efin

emen

t:

392±

10


Pv 1 Pv 2 Pv 3 HC 1 HC 2 HC 3

Pv 1 Pv 2

Pv3

HC 1 HC 2

HC 3

Pv 1 Pv 2

Pv 3

HC 1 HC 2

HC 3

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Up-regulated Down-regulated

HC Pv Pv HC


0

10

20

30

40

50

60

70

80

< 1.5 1.5 - 2.0 2.0 - 3.0 3.0 - 5.0 5.0 - 10 > 10 0

10

20

30

40

50

60

70

80

90

< 1.5 1.5 - 2.0 2.0 - 3.0 3.0 - 5.0 5.0 - 10 > 10

Data are represented as mean ± SEM where (n=3)

Num

ber o

f spo

ts

Fold change

Num

ber o

f spo

ts

Fold change

Down-regulated Up-regulated

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Inte

nsity

Elution time (min)

Your name

Search title

Email

Database(s)

SwissProt NCBInr MSDB

Enzyme

Taxonomy

Fixed modifications Variable modification

Peptide tol. MS/MS tol. Peptide charge Data file Choose

file

Monoisotopic Average

Proteomics [email protected]

Sample protein

Trypsin

Bacterial

Oxidation (M)

1.2 Da Da 0.2

Quantitation

# C13

Data format Instrument

Precursor Start search…

ESI-Q-TOF

LC-MS Data Analysis

Reflector

Detector

LASER

TOF 1 TOF 2

Collision Cell

He He

He He

He He

He He

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Standard: BSA

Serum Proteins: Spot 1 Serum Proteins: Spot 2

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Down-‐regulated proteins

Up-‐regulated proteins

* Haptoglobin precursor (HP)

* Apolipoprotein A-1 (APO A-1)

* Serum albumin precursor (ALB)

* Clusterin precursor (CLU)

* Serum amyloid A (SAA)

* Ceruloplasmin precursor (CP)

* Leucine-rich α-2-

glycoprotein precursor (LRG)

* Alpha-1-antitrypsin precursor

(Alpha-1 protease inhibitor)


•  Few differentially regulated serum proteins identified in this study have not been reported earlier in vivax malaria pathogenesis

•  An important role of serum amyloid A and P, haptoglobin, apolipoprotein A-1 and E proteins elucidated in vivax malaria

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•  Two dimensional electrophoresis can be applied for various applications

•  Case studies –

•  Host response to malaria infection •  Drug treatment to malaria parasite


•  Ray et al. Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response. J Proteomics. 2011. PMID: 22086083

•  Ray et al. Proteomic Investigation of Falciparum and Vivax Malaria for Identification of Surrogate Protein Markers. PLoS ONE 7(8): e41751. doi:10.1371/journal.pone.0041751

•  Herbert BR, Harry JL, Packer NH, Gooley AA, Pedersen SK and Williams KL. What place for polyacrylamide in proteomics? Trends Biotechnol. 2001, 19 (10 Suppl), S3-9.

•  Hanash S. Disease proteomics. Nature 2003, 422, 226-232.

An overview of 2DE technique Applications - nptel.ac.in · Plasmodium malariae Quartan Malaria IIT...

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Transcript of An overview of 2DE technique Applications - nptel.ac.in · Plasmodium malariae Quartan Malaria IIT...