DATA ANALYSIS OF 2DGEL

SUBMITTED BY:PRATEEK KUMARM.Sc BIO-INFORMATICS

INTRODUCTION: Gel Electrophoresis is used to

separate proteins from the mixture of different proteins of different molecular size.

2D Gel Electrophoresis separates the proteins of same molecular size.

It was given by O’Farrell and Klose in 1975.

Firstly, the Serum proteins were separated by using this method.

It separates the proteins based on molecular size as well as charges.

The goal of two-dimensional electrophoresis is to separate and display all gene products present. It is the only method currently available which is capable of simultaneously separating thousands of proteins. The two dimensions of 2D PAGE are –

-iso electric point-size

Therefore, it has unique capability to capture detailed info about protein expression, isoforms, complex formation and post translational modification. e.g.- chemotherapy to cancer cells,

occupational benzene exposure to blood cells.

Progress :-- Chemical or Mass spectrometricanalysis- Immobilized pH gradient (IPG) gels- More sensitive detection procedures- Computer software.

Drawbacks :-

- Poor reproducibility- Limited sample loading

SAMPLE LOADING ON IPG GELS:

BIO-RAD PROTEAN IEF cellAmersham Pharmacia BiotechIPGphor

Multiphor II

DATA ANALYSIS:To study the variations in protein expression

among a series of gels, the gels should be matched together.

Data analysis can be carried out at two different levels-

1- Analyze gels- Study protein expression changes within a set of gels, without taking populations into consideration.

The analytical methods used include scatter plots, descriptive statistics, histograms, and factor analysis.

2- Analyze Classes- Find significant protein expression changes between classes of gels. For this type of analysis, images must be placed in classes and opened in s classes sheet.

The analytical methods used include descriptive statistics per class, histograms, overlapping measure, and statistical tests.

MAJOR STEPS IN GEL ANALYSIS:Data acquisition Gel processing / visualization Spot detection and quantitation Gel matching Gel comparison Data analysis Gel annotation - building a database Link to databases (Internet)

PROCESSPreparation

• Collect samples for 2DE.• The sample was spiked with a

denatured and fluorescently prelabelled protein standard( for accurate alignment of gel images).

Processing

• The standard proteins were selected for their mol size and charges to ensure a standard image that covers the gel as much as possible.

Labeling

• The std images and protein sample were electroblotted from the SDS-PAGE gel to a membrane followed by immunolabelling and visualization by digital camera capture.

Imaging • Sample image-

chloroluminiscent- gel signal image

Imaging • Standard image-

flouroscent- gel standard image

Purpose of images

• Signal image -> proteins to be studied

• Standard image -> image alignment

STAININGSensitive, quantitative and MS-compatible. Comassie brilliant blue R250 staining (30-100

ng) Colloidal comassie blue G250 staining (30-100

ng) Diamine silver stain (1-10 ng) :

gluteraldehyde Silver nitrate stain (1-10 ng) Sypro Ruby fluorescent stain Flamingo fluorescent gel stain.

IDENTIFICATION OF SPOTS:Comparison with a reference gel. Extraction of spots and biochemical analysis.The protein is detected as a strip of

interconnected spots with different sizes and shapes.

The spots change their shape and size with stimulation, as well as increase/decrease in number.

2DE Gel Analysis –what are we looking for?interesting protein spots

-different abundance caused by variation of experimental factor

reliable findings-statistical analysis-based on replicates

Perfect basis for quantitative analysis: complete expression profiles

IN GEL DIGESTION:Spot picking (gel excision) – manual or

mechanical ?

image analysis aspects-speckle artefacts-varying signal intensity-background signal

matching of spots across gels-differences in spot positions-differences in spot patterns-conflicting pair-wise spot matching

missing spots-unique spot matching-statistical significance

2DE Gel Analysis –why a challenge?

IMAGING SOFTWARE:

Amersham Pharmacia BiotechTyphoon

BIO-RADMolecular Imager FX

Imaging

OTHER SOFTWARES ARE: BioNumerics 2DDelta 2DImageMasterMelaniePDQuestProgenesis and REDFIN

MASTER GEL E.coli:

AlignmentAlignment of the signal images is required

to handle spatial offset between gel images, and is achieved by manually aligning all images to a reference image.

To avoid bias from the protein expression in alignment, separate standard gel images are used in the alignment process

NormalizationNormalization of the recorded images is needed

because there will still be some gel to- gel image variability in 2D gel electrophoresis, mainly due to manual preparation and handling of membranes.

The application implements three normalization schemes:

1. The mean normalization-uses the mean pixel value in each image as a normalization scale .

2. The median normalization- uses the median pixel value in each image as the normalization.

3. The Z-score scale normalization- implements a z-score normalization of each pixel based on the mean and the standard deviation of each image.

RESULT-After alignment, the user selects an external variable(e.g. age, height, survival in months) and runs the correlation analysis. This will result in a Spearman rank correlation value, a normalized standard deviation, and a p-value resulting from a correlation t-test or permutation test for each pixel column in the gel stack. For each of these types of values an image is created. Heat map visualization is used to present the results.The correlation measurement was performedby calculating the Spearman rank correlationbetween a chosen external variable (e.g. age, sex, survival in months) and the set of pixels at each pixel coordinate (x, y).

• the t-test for correlated samples, for the simple reason that the two sets of measures in such a situation are arranged in pairs and are thus potentially correlated.• it typically involves situations in which each subject is measured twice, once in condition A, and then again in condition B.• it is very effective in removing the extraneous effects of pre-existing individual differences. 

t-test

Proteomic databases

Collection of data obtained by 2D gels analysis.

It includes:- Annotated images of 2D gels.- Description of identified proteins.

SWISS-2DPAGE: Created in 1993 Collaboration with Central Laboratory of

Clinical Chemistry, University Hospital of Geneva , Swiss Institute of Bioinformatics

36 reference maps– Plasma (27%), E. coli (18%, 8 maps), Mouse(17%, 6 maps)– Human lymphocytes, Staphylococcus aeurus

1265 protein entries 4309 identified spots.

QUERY INTO THE DATABASES:

http://www.expasy.org/ch2d/ Query by keywords Graphical query List of identified proteins per master.

GRAPHICAL QUERY RESULT:

THANK YOU

Spearman rank correlationThe Spearman rank correlation is a

measure of how a change in the external variable corresponds to an increase or decrease in the image pixel intensity.