an overview of 2de technique applications -...
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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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SDS PAGE
2DE DIGE
GEL ANALYSIS
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Isoelectric focusing
IPG strip
Increasing pI
2DE-GEL
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IIT Bombay Proteomics Course NPTEL 5
Gel electrophoresis unit
SDS-Polyacrylamide Gel
Second Dimension: SDS-PAGE
Decreasing Molecular
Weight
Molecular weight
IIT Bombay Proteomics Course NPTEL 6
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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IIT Bombay Proteomics Course NPTEL 7
Gel1 -‐ Control Gel2 -‐ Treatment
IIT Bombay Proteomics Course NPTEL
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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IIT Bombay Proteomics Course NPTEL
• 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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IIT Bombay Proteomics Course NPTEL
• 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
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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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IIT Bombay Proteomics Course NPTEL
• 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
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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
IIT Bombay Proteomics Course NPTEL 24
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
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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
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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
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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
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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
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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
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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
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 proteomics@gmail.com
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)
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• 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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IIT Bombay Proteomics Course NPTEL 41
• Two dimensional electrophoresis can be applied for various applications
• Case studies –
• Host response to malaria infection • Drug treatment to malaria parasite
IIT Bombay Proteomics Course NPTEL
• 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.
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