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Settled Particle Mixture from Previous Step

Settled Particle Mixture from Previous Step

Starting Particle Mixture

Density 3 Solution

Density 2 Solution

Density 1 Solution

Add Particles Let Settle

Add Particles Let Settle

Add Particles Let Settle

Remove Floating Particles

Remove Floating Particles

Remove Floating Particles

Fraction 3 Density 3

Fraction 2 Density 2

Fraction 1 Density 1

Density Based Gradient Extraction

Technology

¾ Results from each group were analyzed statistically.

50.00

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Western Blot of Marker

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45%

Sucros Fraction (w/w)

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Relative % Distribution per Fraction

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Edge 200

Control

Ratio of Marker in Specific Fractions in Control and HF Diet Samples

HF

Data Clustering from Biomarker Analysis

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Collected Fractions

6mm

Sample Rotor

Edge Technology Benefits

Sample Container

29mm

Edge 200 Separation System

of multiple samples

Statistical Analysis

Western Blot

Analysis of Western Blot Showing Relative % Distribution of Biomarker within Control and HF Diet Samples

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tissues

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• Each extraction step takes about 5 minutes.

and can be as low as 100 µl.

• Extraction volume is flexible, user-defined,

• Starting sample volume can be 50 μl to 3.5 ml.

• Fractionation conditions are non-denaturing.

Extraction may start at any density step of interest without the need for going through the whole gradient.

• The recovery yield is > 90%.

and no other instrument is needed for density determination.

• The density of the extraction medium at each step is pre-defined

and does not need any gradient mixer.

•The method DOES NOT need any density gradient medium

• Sample complexity is reduced significantly.

abundance protein isolation and enrichment, biomarker discovery, as well as information on subcellular location.

• Edge Technology provides a powerful, selective sample fractionation method for proteomics analysis, including low

¾ All fractions from each sample were subjected to western analyses using synaptic dysfunction marker p-synapsin I and oxidative stress marker GRP75.

¾ Tissues were homogenized, then fractionated using Edge™ 200 Separation System.

¾ Brains and hearts from each group were collected and snap frozen.

¾ Twenty rats were separated into two groups of 10, one group fed with special high fat diet for 6 weeks and one group fed with regular low fat diet for the same time.

Experiment

Control Tissue

Homogenize

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Western Blot

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Sucrose Fraction (w/w)

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Relative Distribution of P-Synapsin I from Brains of Control Diet Rats Relative % Distribution per Fraction

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60%

C9 C10

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C6 C7

C5

C3 C4

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C1

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Sucrose Fraction (w/w)

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Relative Distribution of P-Synapsin I from Brains of HF Diet Rats Relative % Distribution per Fraction

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T 10

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B

0.96

HF

0.58 1.16

0.68 1.15

1.10

0.69

0.78 1.13

0.58 1.15

0.64 0.80

0.41 0.79

0.52 1.03

0.54 0.93

1.02

0.64

Mean

0.144

0.165

SD

Control

HF

Ratio of p-Synapsin I in Fraction 10% vs. Fraction 40% Control and HF Diet Rat Brains

15%

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25% 30%

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50% 55% 60%

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40% Sucrose Fraction (w/w)

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Relative Distribution of GRP75 from Hearts of Control Diet Rats Relative % Distribution per Fraction

55%

60%

C11

C9 C10

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C1

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Relative Distribution of GRP75 from Hearts of HF Diet Rats Relative % Distribution per Fraction

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1.22 1.55

1.79

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2.09

0.87

1.87

1.28

1.73

1.24

1.44

1.47

1.69

0.83

Ratio of Relative Expression of GRP75 in Frx 20% vs Frx 25% 0.89 1.89

1.18 1.37

0.93 1.69

1.71

1.14

Mean

Conclusions

Control

HF

Ratio of GRP75 in Fraction 20% vs. Fraction 25% Control and HF Diet Rat Hearts

Graphical representation of data above. Error bars indicate mean +/- 1.25 SD.

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Tabulated data from western blot analysis above showing a specific ratio (Frx 20%/ Frx 25%) of GRP75 expression in control and HF diet rat hearts. Also showing mean and SD for each group.

1.40 1.72

Control

Statistical Analysis of GRP75 Distribution Data

• The ratio of marker expression between two specific fractions can be used to determine if the sample is control or HF diet.

• Ratios of marker expressions between two specific fractions within a sample are different for control and HF diet samples, with relatively tight clustering of data for both markers.

• Relative distributions of these markers within fractions of a sample are different between control and HF diet samples.

T11

T9 T10

T8

0.206

0.225

SD

B

www.prospectbiosys.com

Prospect Biosystems, LLC

• Non-genetically identical animals were used in this experiment, and it is expected that results using this method with genetically identical animals will generate tighter clustering of data.

• Because the method relies on ratio between internal fractions of the sample, the method is independent of sample amount.

• The method DOES NOT require internal or external standards.

55%

T6 T7

T5

T3 T4

T2

T1

Western blot analysis of GRP75 from fractionated rat heart samples. A: Relative distributions of GRP75 within fractions of control hearts. B: Relative distributions of GRP75 within fractions of HF diet hearts. Note: Two groups of 11 rats each were used in this study.

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HF

A

10%

Western Blot

Western blots of GRP75 from Edge fractionated rat brains. One sample each of a control and a HF diet rat heart is shown.

HF Heart #2

Control Heart #4

Evaluation of GRP75 in Heart

• Two potential biomarkers, p-synapsin I and GRP75, were evaluated as indicators of synaptic dysfunction and oxidative stress, respectively, using Edge technology and a newly developed statistical analysis method.

Graphical representation of data above. Error bars indicate mean +/- 1 SD.

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Tabulated data from western blot analysis above showing a specific ratio (Frx 10%/ Frx 40%) of p-synapsin I expression in control and HF diet rat brains. Also showing mean and SD for each group.

1.01

Control

Ratio of p-Synapsin I Expression in Frx 10% vs Frx 40%

Statistical Analysis of p-Synapsin I Distribution Data

Western blot analysis of p-Synapsin I from fractionated rat brain samples. A: Relative distributions of p-Synapsin I within fractions of control brains. B: Relative distributions of p-Synapsin I within fractions of HF diet brains.

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Western blots of p-Synapsin I from Edge fractionated rat brains. One sample each of a control and a HF diet rat brain is shown.

HF Brain #3

Control Brain #7

Relative % Expression

HF Tissue

Ratio of Frx "x" /Frx "y"

Evaluation of p-Synapsin I in Brain

Relative % Expression

Work Flow

Relative % Expression

Introduction

Relative % Expression

The identification of biomarkers is an essential element for early prediction of diseases and, in the future, personalized medicine. Biomarker discovery, evaluation and validation are the key steps in biomarker development processes. The recent development of high-throughput proteomics, including high sensitivity mass spectrometry and automation of protein identification, significantly increases the data base of potential biomarkers. However, the evaluation and validation steps of the biomarker development process remain a bottleneck. Edge (Enhanced density gradient extraction) technology, a density based extraction method, provides 1) a simple and reproducible technique for fractionation of tissue homogenates/cell lysates, 2) a unique statistical method for evaluation of potential biomarkers, and 3) the opportunity for generating data for further clinical validation. To demonstrate the utility of Edge technology in biomarker evaluation, rat models of high fat diet (HF) were used in this study, since high fat diet has a profound impact on brain function and cardiovascular risk.

Ratio of Frx 20%/Frx 25%

Wenkui Lan, Thuy Do and Marc J. Horn, Prospect Biosystems, LLC, Newark, NJ

Statistical Demonstration of the Utility of Edge™ Technology in the Evaluation of Biomarkers

Ratio of Frx10%/Frx 40%

Relative % Expression

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