• Case studies 2DE: • Drug treatment in malaria parasite • Plasma proteome analysis of SARS • An overview of DIGE technique • Case study DIGE: • Serum proteome analysis of prostate cancer

(1)

Dr. Sanjeeva Srivastava IIT Bombay

IIT Bombay Proteomics Course NPTEL

•  Case studies 2DE:

•  Drug treatment in malaria parasite

•  Plasma proteome analysis of SARS

•  An overview of DIGE technique

•  Case study DIGE:

•  Serum proteome analysis of prostate cancer

2

(2)

Makanga et al. Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria.

Proteomics. 2005, 5, 1849

Case study-1

•  CoArtem is a combination of artemisinin- derivative artemether with lumefantrine

•  Drug of choice for all cases of non-severe malaria worldwide

•  Artemisinin drug action is mediated specifically through its endoperoxide moiety

4

(3)

IIT Bombay

5

Proteomics Course NPTEL

Plasmodium falciparum Proteome

IIT Bombay

6

Plasmodium falciparum

Late ring stage of Plasmodium

falciparum

Microscopic field showing Pf in its late

ring stage in the synchronised culture

Log

Lag Phase

Log Phase

Death Phase Stationary Phase

Time

Culture of late

ring stage pf

(4)

IIT Bombay

7

Fractionation of the P. falciparum proteome

Synchronised parasites isolated from host erythrocytes Washed and solubilized in a Tris buffer

Tris-insoluble fraction was subjected to two further extractions in urea based lysis buffers

IIT Bombay

8

Protein bands after staining

_

+

Sample wells

Buffe r

Gel

Glass/

Plastic plates

Protein Samples loaded

into sample wells

(5)

IIT Bombay

9

CoArtem = Lumifantrine + Artemether

Reaction with Late ring stage of Plasmodium falciparum

Glycolytic Enzymes

Alterations in Parasite Proteome

IIT Bombay

10

Artemether Lumefantrine

Membrane associated calcium binding protein

Aspartic proteinase (HAP) HSP60, 70, 90

Enolase

Fructose biphosphate aldolase Phophoglycerate kinase

D

U

U U

D U

(6)

IIT Bombay

11

Stress Response

Proteins

ARM LUM

Upregulated Downregulated

Glycolytic Enzymes

ARM LUM

IIT Bombay

12

•  Investigation of alterations to the parasites proteome induced by the two components of CoArtem,

artemether and lumefantrine

•  Established relationship between pharmacologically

relevant concentration and time of exposure for the

two components of CoArtem

(7)

Chen et al. Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and

mass spectrometry. Proc Natl Acad Sci USA. 2004, 101, 17039

Case study-2

IIT Bombay

14

•  Plasma proteome analysis of severe acute respiratory syndrome (SARS)

•  Technique: 2DE and Mass Spectrometry

(8)

IIT Bombay

15

Respiratory Disease

Droplets of saliva or mucus infected with

SARS virus

Membrane Glycoprotein

(M)

Nucleocapsid Phosphoprotein (N)

Spike Glycoprotein

(S)

RNA

SARS Virus

IIT Bombay

16

_

+

Sample wells

Buffe r

Gel

Glass/

Plastic plates Protein Samples loaded into

sample wells Protein bands

after staining

385 Proteins with >2 fold change excised

Trypsin Digestion Trypsin

38 Spots selected for

Protein Identification 22 plasma

samples 4 SARS patients

6 Healthy Patients 6 Healthy Controls

(9)

IIT Bombay

17

IIT Bombay

18

Intensity

Elution time (min)

Your name Search title

Email

Database (s)

SwissProt NCBInr MSDB

Enzyme

Taxonomy Fixed modification s 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 0.2 Da

Quantitation

# C¹³

Data format

Instrument Precursor

Start search…

ESI-Q-TOF

LC-MS Data Analysis

Mass Analyzer #2

Collision

Mass Analyzer #1

Cell Ion

Source

(10)

IIT Bombay

19

Differentially expressed proteins

IIT Bombay

20

SDS-PAGE

PVDF

(11)

IIT Bombay

21

1 2 3 4

44 kDa 22 kDa Non–reducing Conditions

1 2 3 4

Recomb PrxII Normal Patient Sample 1 Patient Sample 2 Recomb PrxII Normal Patient Sample 1 Patient Sample 2

Reducing

IIT Bombay

22

T cell T cell Receptor Viral peptide HLA Class I CD8

Virus

Peroxiredoxin II

(12)

IIT Bombay

23

Acute Phase Proteins in Excess

Indicates bad liver responsible for weak

defense against SARS

SARS Corona Virus Infection

(13)

IIT Bombay

25

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

IIT Bombay

26

Sample mixing and 2-DE

D ir ec ti o n o f m ig ra ti o n ,

Internal standard Sample 1 Sample 2

pH 3

pH 9

Inc re a sing pH D ir ec ti o n o f m ig ra ti o n ,

(14)

DeCyder

Software pH 4 pH 7

pH 4

Increasing pI

pH 7

Cy3 Excita5on

Cy5 Excita5on

Cy2 Excita5on

IIT Bombay

28

HC PC

DeCyder Software

Representative Three dimensional Protein expression profiles

(15)

Your name Search -tle

Email

Database(s) SwissProt NCBInr MSDB

Enzyme

Taxonomy Fixed modiﬁca-ons Variable modiﬁca-on

Pep-de tol. MS/MS tol.

Pep-de charge Data ﬁle Choose ﬁle

Sample protein

Trypsin

Bacterial

Oxida-on (M)

1.2 Da 0.2 Da

Quan-ta-on

# C¹³

Data format

Start search…

ESI-‐Q-‐TOF

LC-‐MS Data Analysis

Intensity

Elu-on -me (min)

Mass Spectra - HC Mass Spectra - PC

Ion Source

(16)

Byrne et al. 2D-DIGE as a Strategy To Identify Serum Markers for the Progression of Prostate Cancer

J Proteome Research. 2009, 8, 942-57

Case study-3

Identiﬁcation of serum markers depicting progression of prostate cancer using DIGE

Prostrate

Cancer DRE

PSA

Biopsy

Prostrate Cancer Serum

Proteomic Analysis

Healthy Human

Healthy Control Serum

Diagnostic Markers for early prostrate cancer

Tissue

Sample Tissue

Sample

(17)

Loading of unpuriﬁed Protein

Mixture Sample

Eﬄuent sample frac5ons

Collected Concentra5on

Detector Deriva5zed

sta5onary phase

Ligand solu5on

Cy3

Cy2

Cy5 2 St an dar d (1 +2 ) 1

Cyanine dyes

Mix

(18)

+

Sa mp wells le

Bu ffer Gel

Glass/

Plastic plates Protein bands

after staining- PC

2-‐D Electrophoresis

SDS-Polyacrylamide Gel

(19)

IIT Bombay

37

Intensity

Elution time (min)

Your name Search title

Email

Database (s) SwissProt

NCBInr MSDB

Enzyme

Taxonomy Fixed modification s Variable modification

Peptide tol. MS/MS tol.

Peptide charge

Data file Choose file

Sample protein

Trypsin

Bacterial

Oxidation (M)

1.2 Da 0.2 Da

Quantitation

# C¹³

Data format

Start search…

ESI-Q-TOF

LC-MS Data Analysis

Mass Analyzer #2

Collision

Mass Analyzer #1

Cell Ion

Source

IIT Bombay

38

DeCyder Progenesis

Pigment epithelium-derived factor Zinc- α 2-glycoprotein

Ficolin 3

Apolipoprotein A-II

U U

D D

U

(20)

Capture Ab Target protein 1⁰Antibody

2ry Antibody

96 well plate

ELISA

PEDF

ZAG

Serum Blocking

reagent

Tissue

Chromogen

(21)

IIT Bombay

41

•  Serum markers reflective of pathological grade and stage are beneficial for identification of appropriate treatment strategies

•  Confirmed differential expression of PEDF and ZAG by various validation techniques

•  PEDF a potential marker of early stage prostate cancer prediction

IIT Bombay

42

•  Two dimensional electrophoresis (2DE and 2D- DIGE) can be applied for various applications

•  Case studies –

•  Drug treatment in malaria parasite

•  Plasma proteome analysis of SARS

•  Serum proteome analysis of prostate cancer

(22)

•  Makanga et al. Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria. Proteomics. 2005, 5, 1849

•  Chen et al. Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass

spectrometry. Proc Natl Acad Sci USA. 2004, 101, 17039

•  Byrne et al. 2D-DIGE as a Strategy To Identify Serum Markers for the Progression of Prostate Cancer. J Proteome Research. 2009, 8, 942-57

•  Issaq H, Veenstra T. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE): advances and perspectives.

Biotechniques. 2008, 44, 697-8, 700.

•  John F. Timms, Rainer Cramer Professor, PROTEOMICS. Difference gel electrophoresis. Volume 8, Issue 23-24, pages 4886–4897, No.

23-24 December 2008.

•  Ray et al. Serum proteome analysis of vivax malaria: An insight into

the disease pathogenesis and host immune response. Journal of

• Case studies 2DE: • Drug treatment in malaria parasite • Plasma proteome analysis of SARS • An overview of DIGE technique • Case study DIGE: • Serum proteome analysis of prostate cancer

Dr. Sanjeeva Srivastava IIT Bombay

Makanga et al. Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria.

Plasmodium falciparum Proteome

Fractionation of the P. falciparum proteome

Sample wells

Glass/

Aspartic proteinase (HAP) HSP60, 70, 90

• Established relationship between pharmacologically

Case study-2

SARS Virus

Sample wells

Glass/

Intensity

Bacterial

ESI-Q-TOF

T cell T cell Receptor Viral peptide HLA Class I CD8

Cyanine dyes

Your name Search -tle

Start search…

ESI-­‐Q-­‐TOF

LC-­‐MS Data Analysis

Mass Spectra - HC Mass Spectra - PC

Prostrate

Mixture Sample

Glass/

Intensity

Bacterial

ESI-Q-TOF

2ry Antibody

96 well plate

• Confirmed differential expression of PEDF and ZAG by various validation techniques

• Makanga et al. Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria. Proteomics. 2005, 5, 1849

Biotechniques. 2008, 44, 697-8, 700.

Proteomics. Volume 75, Issue 10, 6 June 2012, Pages 3063–3080

•  Established relationship between pharmacologically

ESI-‐Q-‐TOF

LC-‐MS Data Analysis

•  Confirmed differential expression of PEDF and ZAG by various validation techniques

•  Makanga et al. Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria. Proteomics. 2005, 5, 1849