2D DIGE Workflow: An Applied Quantitative 2D DIGE Workflow: An Applied Quantitative Proteomics tool to identify molecular signatures for

uro-genital dysfunction in diabetic rat model.

Elizabeth Elizabeth YohannesYohannes, Ph.D. & Chao Yuan, Ph.D., Ph.D. & Chao Yuan, Ph.D.Center for Proteomics & BioinformaticsCenter for Proteomics & Bioinformatics

Case Western Reserve University Case Western Reserve University April 02, 2009April 02, 2009pr , 9pr , 9

Section I

2D DIGE Workflow

Example Center Project Using 2D-DIGE Platform Example Center Project Using 2D DIGE Platform

Section IISection II

2D gel analysis of Myofilament proteins

Outline11stst partpart

An overview protein profiling methods An overview protein profiling methods

Traditional 2-D gel electrophoresis g p

2D DIGE for Quantitative Proteomics

The three main features in 2-D DIGE

P t i P fili M th dsProtein Profiling Methods

Top down p

Traditional 2-D gel Electrophoresis

2-D difference gel electrophoresis (2D DIGE)

Non - two gel separation methods

Protein and antibody arrays

Bottom upBottom up

Multi-dimensional protein identification technology (MudPIT)

Isotope-coded affinity tags

Accurate mass tag based protein profiling

Shotgun proteomics

Two dimensional (2D) gel Electrophoresis:

O’Farrel (J. Biol. Chem. 1975)

Principle:

l 1st charge or Isoelectric point

2nd size (SDS-PAGE)

-

SDS-PAGESubmerge Submerge

IPG in SDSIPG in SDS

+IPGIPG--stripstrip

Figure 1: Schematic diagram showing the different steps in the 2-D Electrophoresis

Gel to Gel variability

Limitations to Traditional 2D gel Electrophoresis

Gel to Gel variability

- Complex image analysis - System variation

- Induced biological changes

Time consuming, labor-intensive and expensive

Protein Visualization

Coomassie Brilliant BlueDetection limits ~ 1μg protein (~20 ng protein with colloidal coomassie blue-G)Variability from destaining, high backgroundPoor linear response (1 order)

Silver Stain Sensitivity: 1-5 ng proteinpoor linear response (1 order)p pless reproducible

TwoTwo--dimensional Difference gel Electrophoresis (2dimensional Difference gel Electrophoresis (2--D DIGE) D DIGE) ÜnÜnlü lü et alet al

Ünlü M, Morgan ME, Minden JS, g ,Carnegie Mellon University

pH >=8.5

proteinproteinH3N

NHS reactive group

proteinprotein

Figure 2: Schematic representation of the labeling reaction-formation of a covalent amide bond between the NHS ester group of CyDye DIGE fl minim l d nd min p f l sin sid f p t in fluor minimal dye and -amino group of lysine residue of a protein.

Test sample Control sample

TwoTwo--dimensional Difference gel Electrophoresis (2dimensional Difference gel Electrophoresis (2--D DIGE) D DIGE) ÜnÜnlü lü et alet al

p p

Fluor A Fluor B

Test Sample Control Sample est Sample ontrol Sample

Mix

2D gel

Image gelg g

Excitation 1 Excitation 2

Image test Image control

Ünlü M, et al Electrophoresis 1997, 18, 2071-2077Figure 3: The original protocol of 2-D DIGE

Gel Image

Figure 4: An overlay of Cy3 and Cy5 images Viswanathan, S, Ünlü, M et al nature protocols 2006, 1, 1351-1358

Dye Cy2 Cy3 Cy5

Table 1: physical properties of cyanine dyes

Molecular weight 550.60 582.77 580.75

Mass added to Protein (Da) 434 466 464

Color of the fluorescence Green Orange Far red f f g

Absorbance max (nm) 489 550 649

Emission max (nm) 506 570 670

Excitation Filter (nm) 480 (30) 540 (25) 620 (30)Excitation Filter (nm) 480 (30) 540 (25) 620 (30)Emission filer (nm) 520 BP 40 580 BP 30 670 BP 30

Amersham Biosciences (GE Healthcare)

Figure 5: Structure of the cyanine dyes

Dye Cy2 Cy3 Cy5

Table 1: physical properties of cyanine dyes

Molecular weight 550.60 582.77 580.75

Mass added to Protein (Da) 434 466 464

Color of the fluorescence Green Orange Far red f f g

Absorbance max (nm) 489 550 649

Emission max (nm) 506 570 670

Excitation Filter (nm) 480 (30) 540 (25) 620 (30)Excitation Filter (nm) 480 (30) 540 (25) 620 (30)Emission filer (nm) 520 BP 40 580 BP 30 670 BP 30

Amersham Biosciences (GE Healthcare)

D l

Table 2: physical properties of cyanine dyesDye Cy2 Cy3 Cy5 Silver

Sensitivity (ng) 1-5 1-5 1-5 1-5Dynamic range > 3.6 > 3.6 > 3.6 1

Quantitative accuracy of 2D DIGE over traditional 2Quantitative accuracy of 2D DIGE over traditional 2--DEDE

1.1. Multiplexing.Multiplexing. Reduce

- spot pattern variation# f G l2. Internal standard sample.

3 Experimental Designs

- # of Gels- decrease $$$$

3. Experimental Designs

1. Multiplexing.

Quantitative accuracy of 2D DIGE over traditional 2-DE

p g

2. Internal standard sample2. Internal standard sample.

3. Experimental Designs Gel number Cy2 Cy3 Cy51 Pooled standard Control 1 Treated 1

Table 2: An example of experimental design for CyDye DIGE fluor minimal

1 Pooled standard Control 1 Treated 12 Pooled standard Treated 2 Control 23 Pooled standard Control 3 Treated 34 Pooled standard Treated 4 Control 44 Pooled standard Treated 4 Control 4

Alban, A et al, Proteomics 2003, 3, 36-44, , , ,

Quantitative accuracy of 2D DIGE over traditional 2-DE

1. Multiplexing.

2 Int n l st nd d s mpl2 Int n l st nd d s mpl2. Internal standard sample2. Internal standard sample.

3. Experimental Design

Advantages

Each sample with in a gel can be p g p gnormalized to internal standard

Protein abundance can be measured as ratio (not volume)

Accurate quantitation and spot statistics

Separation of experimental from Separation of experimental from inherent biological variation

Internal standard (reference) sample

Without Internal Standard

Gel 1

Without Internal Standard

Without Internal Standard

3

4

(Log

Gel 20

1

23

1 2 3 4

l

Spot

volum

e sc

ale)

Sample 1 Cy3 Sample 2 Cy5

With Internal Standard

Sample

Sample 3 Cy3 Sample 4 Cy5

Gel 1With Internal Standard

3

4

e (L

og

Gel 2 0

1

23

1 2 3 4

SampleSp

ot v

olum

esc

ale)Standard Cy2 Sample 1 Cy3 Sample 2 Cy5

Figure 6: Comparison of gel electrophoresis with and with out internal standard.

Sample

Standard Cy2 Sample 3 Cy3 Sample 4 Cy5

image imageBatch ProcessorBatch Processor

image image.XML .XML

DIADIA BVABVA XML ToolboxXML Toolbox

f l

Image loaderImage loader

.DIA file .BVA file

EDAEDA

Figure 7: Scheme showing the different modules and image analysis workflow in the DeCyder software DeCyder software

Green (532)

Gel 1 Gel 2

Green (532)

Cy3 Image

Cy3:Cy2

Cy3 Image

Red (633)In gel co-detection

Red (633)

Bl (488)

Cy5 Image

detection

Cy5:Cy2

Bl (488)

Cy5 Image

Average Ratio =Cy3/Cy2C 5/C 2

Blue (488) Blue (488)

Average Ratio Cy5/Cy2Cy2 Image

Master

Cy2 Image

BVA Cross Gel

Figure 8: Scheme showing spot co-detection on images from a single gel in the DeCyder DIA module and protein difference ratios and statistics between gels in the DeCyder BVA module

Quantitative accuracy of 2D DIGE over traditional 2-DE

1. Multiplexing.

2. Internal standards (reference) sample.

3 E i t l D iE i t l D i3. Experimental DesignsExperimental Designs

Table 3: Experimental design 1 (Total 8 gels)

Gel Cy3 Cy5 Cy21 C1_1W D1_1W Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M2 D2 1W C2 1W P l d l (C1 4 1W D1 4 1W C1 4 2M D1 4 2M

Table 3 Experimental design 1 (Total 8 gels)

2 D2_1W C2_1W Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M3 C3_1W D3_1W Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M4 D4_1W C4_1W Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M5 C1 2M D1 2M Pooled sample (C1-4 1W + D1-4 1W + C1-4 2M +D1-4 2M5 C1_2M D1_2M Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M6 D2_2M C2_2M Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M7 C3_2M D3_2M Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M

8 D4 2M C4 2M Pooled sample (C1 4 1W + D1 4 1W + C1 4 2M +D1 4 2M8 D4_2M C4_2M Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M

Quantitative accuracy of 2D DIGE over traditional 2D

1 M lti l i1. Multiplexing.

2. Internal standards (reference) sample.

3. Experimental DesignsExperimental Designs3. Exper mental Des gnsExper mental Des gns

Gel Cy3 Cy5 Cy2

Table 4: Experimental design 2 (Total 20 gels)

Gel Cy3 Cy5 Cy2Schizophrenia white mater (10 samples)

Gels 1-5 Schizophrenia Schizophrenia St. pool of sample 1-40

Control white mater (10 samples)

Gels 6-10 Control Control St. pool of sample 1-40samples)Schizophrenia grey mater (10 samples)

Gels 11-15 Schizophrenia Schizophrenia St. pool of sample 1-40

Control grey mater (10 Gels 16-20 Control Control St. pool of sample 1-40samples)

sample size for 2-D DIGE Experiment

Proper sample size is Important

If too large

unnecessary involvement of extra subjects (samples) unnecessary involvement of extra subjects (samples)

Increase $$$$$

If too small:

Unable to detect important differences

If not done properly: Fail to get funding, publication

n = 2(Z/2 + Z)22

(µ1-µ2)2

http://hedwig.mgh.harvard.edu/sample_size/js/js_parallel_quant.html

significance level=0.05

1

1.2

0.6

0.8

ower change 50%

change 30%

0.2

0.4

po

g %change 20%

00 10 20 30 40 50 60 70

sample size

Figure 9: Relationship between power and number of replicates in detecting various fold changes when the variance encompasses 75% of the spots

sa p e s e

various fold changes when the variance encompasses 75% of the spots.

Limitations to 2-D DIGE

2D DIGE does not represent true global technique.

V cidic b sic p t ins Very acidic or basic proteins

Excessively large or small proteinsy g p

Membrane proteins

Low abundant proteins

Dynamic range 2-D DIGE 103-104Dynamic range 2-D DIGE 10 -10

Single cell or individual cell type : low abundant (100 copies), most abundant proteins (106)

Target identification for Diabetes Mellitus Associated Target identification for Diabetes Mellitus Associated g f fg f fUrogenetal Dysfunction.Urogenetal Dysfunction.

In collaboration with Dr. Kelvin Davies (Albert Einstein College of Medicine) In collaboration with Dr. Kelvin Davies (Albert Einstein College of Medicine) & Dr. George Christ (Wake Forest University School of Medicine)& Dr. George Christ (Wake Forest University School of Medicine)

OutlineI. IntroductionI. Introduction

Diabetes and its complicationsDiabetes and its complications

II. Objectives II. Objectives

II Proteomics Approach II Proteomics Approach II. Proteomics Approach II. Proteomics Approach 2D2D--DIGE/MSDIGE/MS

MetaCoreMetaCoreTMTM, Validation Western blotting , Validation Western blotting

IV. Summary of resultsIV. Summary of results

V. Acknowledgement V. Acknowledgement

Diabetes mellitusDi b t llitDiabetes mellitus:• Group of metabolic diseases• Hyperglycemia Diabetes mellitus means “sweet urine”

Type I (= insulin-dependent diabetes = juvenile onset diabetes)– Caused by destruction of the B cellsy– Generally appears in childhood– Absolutely dependent on insulin replacement

Type II (= insulin-independent diabetes = adult onset diabetes)Type II (= insulin independent diabetes = adult onset diabetes)– Caused by target cell resistance to insulin (InsR decreased,

signaling defect)

– Obesity appears to reduce the number of insulin receptors

l b d d l– Mostly appears in obese individuals– Can be treated with oral hypoglycemic drugs

Diabetes mellitus

Pancreas:Pancreas:• Islets of Langerhans: site of

hormone production– (alpha) cells – produce (alpha) cells produce

Glucagon– (beta) cells – produce Insulin– (delta) cells – produce (delta) cells produce

Somatostatin

Insulin and Glucagon are the major Insulin and Glucagon are the major regulators of blood glucoseFigure 1. Anatomy of the middle digestive tract.

Source: © MedicineNet, Inc.

Diabetes mellitusDi b t llitDiabetes mellitus:• Group of metabolic diseases• Hyperglycemia Diabetes mellitus means “sweet urine”

Type I (= insulin-dependent diabetes = juvenile onset diabetes)– Caused by destruction of the B cellsy– Generally appears in childhood– Absolutely dependent on insulin replacement

Type II (= insulin-independent diabetes = adult onset diabetes)Type II (= insulin independent diabetes = adult onset diabetes)– Caused by target cell resistance to insulin (InsR decreased,

signaling defect)

– Obesity appears to reduce the number of insulin receptors

– Mostly appears in obese individualsy pp– Can be treated with oral hypoglycemic drugs

C li tiDiabetes mellitus

Complications:• Short-term

– Hyperglycemia (hypoglycemia)Hyperglycemia, (hypoglycemia)– Ketoacidosis

• Long-term– Disruptions in blood flow => Cardiovascular

complications => Amputations– Retinopathy – blindness– Nephropathy – primary cause of morbidity and

mortalitymortality– Neuropathy – nerve damage–– Bladder dysfunction (Yohannes Bladder dysfunction (Yohannes et alet al MCP MCP

2008 7 12702008 7 1270 85)85)2008,7:12702008,7:1270--85)85)–– Erectile dysfunction (impotence)Erectile dysfunction (impotence)

Erectile dysfunction (ED)Risk Factors:

- psychological => Stress,

- damages => nerves, blood vesicles, and/or smooth muscles.

Men with Diabetes

- 75% (10 to 15 years earlier)- 75% (10 to 15 years earlier)

- worst DSHRQL => Depression, loss of self-esteem, & poor self-image

- Less responsive to pharmacological therapies

P d - Progressive disease

Altered organ Function Further Disease

Compensation & DecompensationOnset Development

Altered organ Function Further Disease Progression

STZ 10 Days

2 Months 4 Months

Figure 2. Schematic representation of the overall duration of the disease in STZ-induced diabetic rat model.

N i d (I i i i d l d i )- No systematic study (Initiation, development and progression)

Objectives To delineate the proteome changes: during the initiation, and

development of diabetes-related ED.

- Using 2D-DIGE/MS platform

T b t i t i i t ti t k di t th ibl To probe protein-protein interaction networks: predict the possible pathways => activated or deactivated

To identify transcriptional factors and relatively low-abundant proteins: Not identified by 2D-DIGE/MS => further analyzed the 2D-DIGE data => MetaCoreTM pathway analysis tools.

To validate 2D-DIGE/MS and MetaCoreTM pathway for specific expression changes by western blotting. p g y g

Experimental Animal Model and induction of diabetes:Animal Model and induction of diabetes:

Animal Models (8-10 weeks F-344 Rat)

Diabetes Control 35 mg/kg body weight STZ vehicle (Citrate buffer) 300 /dl 140 /dl> 300 mg/dl < 140 mg/dl

Erectile responses:

Intracavernosal pressure (ICP) in response to cavernous nerve stimulant => For both

1 week and 2 months diabetes and age much controls

Non-Diabetic Diabetic Diabeticsure

Non D abet cOne Week Two Month

0.6

0.7

Erection when ICP/BPlood

Pre

ss

0.4

0.5

*

*

Erection when ICP/BP>0.55

essu

re/

BlIC

P/BP

)

0.2

0.3

rpor

al P

re (I

0

0.1

0 0.75 4 0 0.75 4 0 0.75 4

Intr

acor

0 0.75 4 0 0.75 4 0 0.75 4

mA (stimulation of cavernous nerve)

Figure 3. Erectile function in non-diabetic rats compared to rats with one weekd 2 th f di b t Th l t ti ti ll i ifi t (* P 0 05 d t and 2 months of diabetes. The values are statistically significant (*= P<0.05 compared to

non-diabetic).

Tissue collection: - sacrificed - penile dissected - corpora cavernous smooth muscle (nitrogen p ( g

flash frozen and stored at - 800C) => 2D-DIGE/MS, Verification

transverse section

Figure 4. The transverse section of penile. Tom F. Lue, The new England Journal of Medicine 324, 1802, 2007

Test hypothesis: Experimental

Time or treatment significantly changes the dependent variable (Protein expression)

There are interaction effects between the two factors (time and treatment)

Table 1. Experimental design. Gel Cy3 Cy5 Cy21 C1 1W D1 1W P l d l (C1 4 1W D1 4 1W C1 4 2M D1 4 2M1 C1_1W D1_1W Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M2 D2_1W C2_1W Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M3 C3_1W D3_1W Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M4 D4 1W C4 1W Pooled sample (C1-4 1W + D1-4 1W + C1-4 2M +D1-4 2M4 D4_1W C4_1W Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M5 C1_2M D1_2M Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M6 D2_2M C2_2M Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M7 C3_2M D3_2M Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2M_ _ p ( _ _ _ _

8 D4_2M C4_2M Pooled sample (C1-4_1W + D1-4_1W + C1-4_2M +D1-4_2MControl_1week (C_1W), Diabetes_1week (D_1W, Control_8-10_weeks (C_2M), Diabetes_8-10_ weeks (D_2M)

cy3 cy5cy2

MIX

VRatio 1.5 or -1.5with P <= 0.05

2D E

3-mode scanning Typhoon

Pick list

Typhoon

DeCyder 2-D Software

Excised gel plug

I l Di tDIA, BVA, & EDA

In-gel Digest

Protein ID/MALDI

Figure 5. An overview of the different steps involved in the 2D-DIGE

Or LC-MS => 57

Statistical outcome

Treatment(Diabetes)

Time Both Interaction effect

# of spots 51 19 100 21

nce

0.5

0.4

0.3

0.2

Protein: 114

Stan

dard

Abu

nda

0.1

0

-0.1

Average ratio: -2.69

Student's t-test:

Log -0.2

-0.3

-0.4

-0.5

One-way ANOVA: 4.68E-6

Two-way ANOVA: 2.78E-6 Time 6.68E-4 treated0 0105 Interaction 0.5

Experiment groups

1 2 3 4

0.0105 Interaction

C_2M

D_2Mce

C_1W

D 1W.7%

of v

aria

nc

D_1W

PC2:

9

PC1: 71.1% of variance

Figure 6. Principal component analysis (PCA) of the proteins mediated by STZ-induced diabetes. The protein expression profiles of experimental groups were visualized in two-dimensional Euclidian space. The PCA, distinctly clustered the 15 individual samples into four experimental groups (C-1W = 1 week control, D-1W = 1 week diabetes, C-2M = two months control, and D-2M = two months diabetes).

0.7

0.6

0.5

0.4

Proteins (Score Plot)ADiabetesControl2MControl Diabetes1W

CPC

2

0.3

0.2

0.1

0

-0.1

-0.2

-0.3

-0 40.4

-0.5

-0.6

-0.7

-0.8

-0.9

-1

PC110-1-2

116115

D0.5

0.4

0.3

Spot 115

Average ratio: 2:1 = -2.94, 4:3 = -7.79PC1

BSt

anda

rd A

bund

ance

0.2

0.1

0

-0.1

-0.2

-0.3

One way ANOVA: 9.28E-09

Two way ANOVA 5.17E-07 Time

9.27E-05 treated

0.289 Interaction

Group 1 Group 2 Value

Log

S -0.4

-0.5

-0.6

-0.7

-0.8

-0.9

-1

1 2 1.90E-03

1 3 2.90E-05

1 4 8.88E-07

2 3 0,0428

2 4 2 09E 04

Control

Figure 7: Differential abundance spot. Principal component analysis of 108 spot features that have statistical significant (ANOVA p<0.05) changes in abundance and are present in all gels (A). The black rectangle on the

1

Experiment groups

1 2 3 4

2 4 2.09E-04

3 4 0.0245Diabetes

gel image shows the region on the gels where the potential outlier spots (115 and 116) were situated. Two magnified views of this region showing control and diabetes images (B). 3D images of spot feature 115 showing the 3 fold decreases at one week time point and 7.5 fold decreases at two months time point in abundance (C1 and C2 respectively). Graphical representation of the standardized log abundance data obtained for spot feature 115 (D).

Figure 8: 2-D gel image, showing the pick location of differential expressed proteins.

Table 2: Protein profile in an STZ-induced rat corpora smooth muscle

Pos. Gene name

Protein identities Accession number

MW (KDa), pI1

Log10(Average ratio)2

2/1 4/3 3/1 4/2

Table 2: Protein profile in an STZ induced rat corpora smooth muscle

6 Apoa1 Apolipoprotein A-I precursor P04639 30.1, 5.5 2.89 3.39 1.01 1.18

7 Apoa4 Apolipoprotein A-IV P02651 44.5, 4.98 2.24 2.03 1.21 1.1

8 Apoe Apolipoprotein E precursor P02650 35.8, 5.2 -1.97 -2.0 -2.21 -1.26

17 Col14a1 Collagen, type XIV, alpha 1 gi109480777 193.22, 4.9 -1.11 -1.68 -1.11 -1.69

18 Col1a1 Collagen, type 1, alpha 1 P02454 137.86, 5.7 -1.68 -2.02 -2.57 -3.12

19 Col1a2 Collagen, type 1, alpha 2 P02466 129.48, 9.6 -1.96 -2.04 -2.62 -1.81

20 Col6a2 Collagen, type 6, alpha 2 Q5EB88 109.5, 6.2 -1.52 -2.02 -1.7 -1.75

51 Hsp47 heat shock protein P29457 46.6, 9.2 -2.94 -7.79 -6.09 - 4.17

36 Igc anti-NGF antibody light-chain

gi4096754 23.80, 5.9 1.86 2.02 1.66 1.69

Hipp, JD et al (2007) BJU Int 99, 418-430 and Sullivan, CJ et al(2005)Physiological genomics 23, 192-205

Spot 155 (Igc)

0.3

0.25

0.2

0.15

Average ratio: 2:1 = 1.86, 4:3 = 2.02

One way ANOVA: 3.68E-05

Two way ANOVA 1.27E-05 Time

dard

Abu

ndan

ce

0.15

0.1

0.05

0

1.95E-03 Treated

0.2 Interaction

Log

Stan -0.05

-0.1

-0.15

-0.2

Group 1 Group 2 Value

1 2 0.013

1 3 2.88E-04

-0.25

-0.3

Experiment groups1 2 3 4

1 4 3.73E-05

2 3 0,0592

2 4 3.02E-03

2-D DIGE expression profile of Igc 3 4 2.32E-4

Diabetic rat models => in Tissue NGF level in the bladder and Lumbosacral dorsal root ganglia (DRG) <=> the progression of diabetic cystopathy

FIG. 9. NGF levels measured by enzyme-linked immunosorbent assay in the bladder (A) and L6 DRG (B) of normal rats (n = 12), untreated diabetic rats 12 weeks after STZ injection

FIG. 10. Micturition patterns in a metabolic cage study to evaluate the efficacy of HSV vector–mediated NGF delivery to the bladder A: (DM12W), diabetic rats with SHZ (HSV-1 without NGF)

injection (SHZ, n = 6), and diabetic rats with SLN (HSV-1with NGF) injection (SLN, n = 8). Virus vectors were injected 8 weeks after diabetes induction, and NGF levels were measured 4 weeks after virus injection. Note that reduced NGF levels in the bladder and L6 DRG were significantly elevated in diabetic rats with SLN injection compared with

mediated NGF delivery to the bladder. A: Representative traces of voided urine volume plotted against time in normal rats (upper trace) and diabetic rats (middle trace: diabetic rats with SHZ control vector injection; lower trace: diabetic rats injected with SLN). B: Averaged voided volume per micturition (normal rats: n = 9; SHZ: n = 10; SLN: n = 11). **P < elevated in diabetic rats with SLN injection compared with

untreated diabetic rats and diabetic rats with SHZ injection (bladder: P < 0.01; L6 DRG: P < 0.05). *P < 0.05, **P < 0.01. prot., protein.

Sasaki, K., (2004) Diabetes 53, 2723-2730

( ; ; )0.01, *P < 0.05.

Activation effectActivation effect

h b ffh b ffOverexpressed proteinOverexpressed protein

Figure 11: Protein networks associated with differential expressed proteins in response to STZ-induced diabetes.

Inhibition effectInhibition effect

Unspecified Unspecified Underexpressed proteinUnderexpressed protein

Verification of 2D-DIGE/MS & MetaCoreTM Results

Hsp47 47

0.5

0.4

0.3

BA

β-actin 42

Hsp 7 47

ard

Abu

ndan

ce

0.2

0.1

0

-0.1

-0.2

0 3

Log

Stan

da -0.3

-0.4

-0.5

-0.6

-0.7

0 8

C-1

W-1

C-1

W-2

D-1

W-1

D-1

W-2

C-2

M-1

C-2

M-2

D-2

M-1

D-2

M-2

-0.8

-0.9

-1

Experiment groups

1 2 3 4

Figure 12: A) Conformational immunoblots for Hsp47. B)2-D DIGE expression profile of Hsp47 p p

Verification of 2D-DIGE/MS & MetaCoreTM Results

p53 53C

-1W

-1

C-1

W-2

D-1

W-1

D-1

W-2

C-2

M-1

C-2

M-2

D-2

M-1

D-2

M-2

β-actin 42

C C D D C D

HDAC1 62

β-actin 42

1W-1

1W-2

1W-1

1W-2

2M-1

2M-2

2M-1

2M-2

Figure 13: Conformational immunoblots for p53 and HDAC1, that were hypothetically id ntifi d b n t k n l sis E h l n is l d d ith s mpl f m ind p nd nt

C-1

C-1

D-1

D-1 C-2

C-2 D-2

D-2

identified by network analysis. Each lane is loaded with a sample from independent biological replicate (n = 2/experimental group).

Summary

STZ-ID significantly altered protein expression in corpora smooth muscle.

- Decreased the expression of different isoforms of collages which are - Decreased the expression of different isoforms of collages, which are precursor to fibrils forming collagen type 1, hsp47 that assists and mediates the proper folding of procollagen, type I, alpha 1 and procollagen, type I, alpha 2 and proteins involved in muscle remodeling (eg. LIM protein).

- Increased the proteins involved in oxidative stress (eg. Glutathione peroxidase 3), protein that neutralize the biological activity of nerve growth factor (eg. Anti-NGF), and proteins involved in inflammatory response (eg Fga, Fgb, Fgg, ApoA1, ApoA4, C3, and C5) proteins that suppresses and induced apoptosis (HSCO & p53 respectively) .

• Our study reports novel proteins that may contribute to y p p ydiabetic-dependant development ED.

• Used to develop novel diagnostic, preventative or therapeutic strategies strategies.

• also provide hypotheses that can be tested by future studies.

Acknowledgement Acknowledgement

•• Mark Chance (Mentor) Mark Chance (Mentor) •• Kelvin Davies (Collaborator)Kelvin Davies (Collaborator)•• Jinsook Chang (Collaborator)Jinsook Chang (Collaborator)

•• Janna Kiselar (LCJanna Kiselar (LC--MS)MS)•• Serguei Ilchenko (LCSerguei Ilchenko (LC--MS)MS)•• Giri Gokulrangan (LCGiri Gokulrangan (LC MS)MS)•• Jinsook Chang (Collaborator)Jinsook Chang (Collaborator) •• Giri Gokulrangan (LCGiri Gokulrangan (LC--MS)MS)& Katy Lundberg (MALDI)& Katy Lundberg (MALDI)

•• Hong Zhao (2Hong Zhao (2--D DIGE)D DIGE)

Thanks Everybody in the Center Thanks Everybody in the Center

2D Gel Analysis of Myofilament Proteins

Chao Yuan

Myofilament ProteinsMyofilament Proteins

• Myofilament proteins are responsible for• Myofilament proteins are responsible for muscle contraction and relaxation.

• In heart, this process is Ca2+ regulated and modulated by phosphorylation of key regulatory myofilament proteins.

50

Schematic Diagram of Myofilament Proteins g y

Contraction

TnTTnT TnCTnC TnITnIActinActinCa2+

M BP CTmTm MHCMHC

MyBP-C MLCMLC

Titin

Phosphorylatable proteins are indicated in red

51

Phosphorylatable proteins are indicated in red

OutlineOutline

• Standard 2D protocolStandard 2D protocol• Special 2D protocols for

Ph h t i– Phosphoproteins– Large proteins– Basic proteins– Proteins with close pI points– Proteins with close molecular weights (MW)

Standard 2D Protocol-IEF-MaterialStandard 2D Protocol IEF Material

• IEF buffer– Urea/thiourea (non-ionic detergent)– CHAPS (zwitterionic detergent)

A h l t (P t t i i it ti )– Ampholyte (Prevent protein precipitation)– DTT (Prevent protein oxidation)– BPB (IEF Progression Indicator)BPB (IEF Progression Indicator)

• Maximum detergent strength/minimum ionic strength (no ionic detergents no salt)ionic detergents, no salt)

Standard 2D Protocol-IEF-Protocol

+ ‐• 50 volts overnight (Rehydration)• 50-250 volts in 15 min• 250-10K volts in 3 hours (for 24 cm strips)• 10K volts for 40K volt/hours• Current limit: 50 mA/Gel• Total time: 22~30 hours (24 cm strips)

Voltage Current Resistance Conductivity

Standard 2D Protocol-2nd DimensionStandard 2D Protocol 2 Dimension

Equilibration buffer 1Urea (6M)l l ( )

DTT (1%)

Equilibration buffer 2

Glycerol (30%)SDS (2%) IAA (2.5%)

2DE of Myofilament Proteins2DE of Myofilament ProteinspI (3 to 11, 18 cm)

MyBP-C

MWMWMHC

220KDa

MyBP C

A ti T T

Tm

Actin TnT

TnI(pI 9.5)

MLC1

56MLC2

2D DIGE2D-DIGE

WT Mouse Heart PKC TG Heart

C 2 C 5Cy2 Cy5

Mix/2D Gel

Imaging & Analysis

57

2D-DIGE of Mouse Heart Proteins

MyBP-CGreen: control yGreen: control

Red: PKC TG

Quantification

18 17161514 13 12 11 10 9 8 7 6 5 4 3 2 1

200MyBP-C total protein change: 1.14%

100

150

0

50

123456789101112131415161718(%)

-100

-50

Co detection of Phospho & total ProteinCo-detection of Phospho- & total-Protein

Label Proteins with Cy2/Cy5

2D-Gel analysis

ProQ Diamond Stain

Total Protein: Cy2/Cy5Phospho-Protein: ProQ

60

MCo-detection of Phospho- & total-

Protein

Green: WTRed: TGBlue: ProQQ

Estimation of Phosphorylation Degree (#) for each spot

18 17161514 13 12 11 10 9 8 7 6 5 4 3 2 1

5

ProQ/(Cy2+Cy5); Compare with Spot 3

3

4

1

2

0123456789101112131415161718

Confirmation of Phosphorylation by Western

PVDFNeonatal

Adult

WesternPh h PKCPhospho-PKCmotif Antibody

63

Novel Phosphorylation SitesNovel Phosphorylation Sites

I (PKA)I (PKA)

II (CamK) III (PKA/PKC) III* IVII*

Distribution of IdentifiedDistribution of Identified Phosphorylation Sites

pH 6                                                                                                          6.7

Pro-Q9 8 7 6 5 4 3 2 1

--IIIIIII

IIII*III*

IIII*III*

IIII*III*IV

II

IV

MyBP-C has 5 unphosphorylated spotsMyBP C has 5 unphosphorylated spots

Before CommassieBefore

After Alkaline

CommassieMyomesin

Phosphotase treatment

Commassie

Pro‐QAfter

Some sites are resistant to AP treatment

Special 2D Protocol for MyBP-CSpecial 2D Protocol for MyBP C

Standard Protocol

150 KDa

Special Protocol 150 KDa

Special Protocol for MyBP-CSpecial Protocol for MyBP C

• Sequential IEF: After 1st IEF excise gelSequential IEF: After 1 IEF, excise gel region corresponding to MyBP-C, and perform a 2nd IEF at a higher than normalperform a 2 IEF at a higher than normal voltage.

• Use 5% SDS instead of 2% SDS in• Use 5% SDS, instead of 2% SDS, in equilibration buffer.U 4 12% di t SDS PAGE l• Use 4-12% gradient SDS-PAGE gel.

3 pI 11Detection of Phosphorylated Myofilament Proteins with ProQ

p

20011697

MWMyBP-C ProQ/WT/PKC

664523

18

TnI

Tm

MLC2TnT

18Tm MLC2 TnT TnIProQ

ProQ

MyBP-C

P U P2 P1 U P2 P1 UQ/Cy2/5

Q

Cy2/Cy5

Q

Cy2/Cy5

69Q/Cy2/5

Q/Cy2/5

Special Protocol for TnI (pI 9 5)Special Protocol for TnI (pI 9.5)

Standard Protocol

Special Protocol

7      6     5      4 3 2 1

Special Protocol for TnISpecial Protocol for TnI

H i t l t ki f b i t i i d t• Horizontal streaking of basic proteins is due to protein (cystein) oxidation.

• Cysteinyl oxidation is due to lack of DTT• Cysteinyl oxidation is due to lack of DTT.

• DTT can be depleted during IEF, especially from basic end because it is a weak acidend, because it is a weak acid. 

• A so called “destreak reagent” (HED) did not improve 2D gel resolution of TnI2D gel resolution of TnI.

DTTHED

Special Protocol for TnI

• Perform IEF as usual

Special Protocol for TnI

Perform IEF as usual.• Shortly before the end of IEF, add DTT

(3% 5%) soaked paper wick to the basic(3%~5%) soaked paper wick to the basic end of the gel.C ti IEF f 15 20 i t• Continue IEF for 15~20 minutes.

2DE of Proteins with Close pI Values2DE of Proteins with Close pI Values

Relative Focusing Power of IPG Strip

2DE of Proteins with Close pI Values2DE of Proteins with Close pI Values

24 cm, 3‐10

4.5 5.5

TM NTG

TMTG

T T

pTMNTG

pTMTG

TnT4

MLC-2P1P2

TnT3PTnT3 PTnT4

18 cm, 4.5‐5.5

1DE of Proteins with Close MW1DE of Proteins with Close MW• Standard 1D Gel Recipep

– 10~12% gel– 29:1 (acrylamide:bisacrylamide)

• TnT isoforms (MW; 40 Kda; Difference: 3 aa)• TnT isoforms (MW; 40 Kda; Difference: 3 aa)– 14% gel– 200:1 (acrylamide:bisacrylamide)– 5% Glycerol

• α- and β-MHC (MW 220 KDa; Difference: 0.4 KDa))– 6% gel– 50:1 (acrylamide: DATD)

10% Glycerol– 10% Glycerol

SummarySummary

• Overview of 2D Gel and DIGEOverview of 2D Gel and DIGE• 2DE of Phosphorylation

2DE f l t i• 2DE of large proteins• 2DE of basic proteins• 2DE of proteins with close pI and MW

values

Acknowledgements/PublicationsAcknowledgements/Publications1. Yuan C, Ravi R, Murphy AM. Discovery of disease-induced post-translational

difi ti i di t til t i C O i M l Th 2005 7 234 9modifications in cardiac contractile proteins. Curr Opin Mol Ther 2005;7:234-9.2. Yuan C, Guo Y, Ravi R, et al. Myosin binding protein C is differentially

phosphorylated upon myocardial stunning in canine and rat hearts-- evidence for novel phosphorylation sites. Proteomics 2006;6:4176-86.

3. Xiao L, Zhao Q, Du Y, Yuan C, Walker LA, Solaro R.J., and Buttrick P. PKCε increases phosphorylation of the cardiac myosin binding protein C at Serine 302 both in vitro and in vivo 2007 Biochemistry, 46,7054-61

4 Yuan C Sheng Q Tang H Li Y Zeng R Solaro RJ Quantitative Comparison of4. Yuan C, Sheng Q, Tang H, Li Y, Zeng R, Solaro RJ. Quantitative Comparison of Sarcomeric Phospho-Proteomes of Neonatal and Adult Rat Hearts. Am J Physiol Heart Circ Physiol 2008; 295, 647-56

5. Yuan C. Solaro R.J. Myofilament proteins: from cardiac disorder to proteomic changes Proteomics Clinical Application 2008; 2:788 99changes. Proteomics-Clinical Application 2008; 2:788-99

6. Warren CM, Arteaga GM, Rajan S, Ahmed RP, Wieczorek DF, Solaro RJ. Use of 2-D DIGE analysis reveals altered phosphorylation in a tropomyosin mutant (Glu54Lys) linked to dilated cardiomyopathy. Proteomics. 2008 Jan;8(1):100-5

7. Biesiadecki BJ, Elder BD, Yu ZB, Jin JP. Cardiac troponin T variants produced by aberrant splicing of multiple exons in animals with high instances of dilated cardiomyopathy. J Biol Chem 2002;277:50275-85.

The Third Quantitative Proteomic A h A tib d AApproach-Antibody Array

S l 1 S l 2Sample1 Sample2

Cy3 Cy5y y

CloneTechCloneTechAb Array 500

78

Ab Array-Preliminary Results (n=1)

Cy3: PGDH‐‐ Crypt, Cy5: PGDH++ CryptCy3: PGDH Crypt, Cy5: PGDH++ CryptReverse labeling was also performed

79Yellow means no change, red or green means protein changes.

Three Proteomic ApproachesThree Proteomic Approaches

2D Gel Mass Spectrometry

Proteomic Changes

Antibody Array

81

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