Version 1.0

Indian Institute of Technology Bombay, India | Bhushan N Kharbikar

PROTEOMICA DEMYSTIFYING PROTEINS

Software User’s Manual

Version 1.0

Copyright © 2013

Bhushan N Kharbikar, Proteomics Lab

Department of Biosciences and Bioengineering

Indian Institute of Technology Bombay

CONTENT

PROTEOMICA 3

INTRODUCTION 3

WHAT IS PROTEOMICA? 4

FIRST TIME USER EXPERIENCES (FTUE) 5

CONFIGURING ENVIRONMENT 5

LAUNCHING PROTEOMICA 5

PRINCIPAL GRAPHICAL USER INTERFACE 8

KNOW THE CONTROLS 9

IMAGE DOMAIN BASED APPROACH 9

DESCRIBING PROTEOMICA GUI 10

WORKFLOW 10

COMMAND BUTTONS 12

SAMPLE GEL ANALYSIS USING PROTEOMICA 15

SET UP AN EXPERIMENT 15

CREATE MATCHSET 15

PRE-PROCESSING OF IMAGES 17

GEL IMAGE MATCHING 20

MATCH AND ALIGN 20

GLOBAL GEL 24

SPOT DETECTION 26

SEGMENT 26

QUANTIFY 32

RESULT AND UTILITY TOOLS 34

STATISTICS 34

3D VIEW VISUALISATION TOOL 39

PROFILE PLOT 44

HELP 50

EXIT 50

REFERENCES 51

Proteomica

Introduction

The processing and analysis of 2 DE gel images is crucial step in proteomics workflow and

have substantial impact on final qualitative and quantitative outcome. Due to large

complexity in protein patterns in gel images, its analysis is very subtle and tedious hence

computer assisted automated processing and analysis is inevitable.

While 2 DE technique for study of proteome is matured over last 3-4 decades, computer

assisted automated techniques are still evolving, even after their introduction between late

70s to early 80s.

Majority of research publications and commercial packages emerged in due course of time

offers varied approaches towards implementation of image processing workflows for 2 DE

gel images. More robust algorithms comes up which effectively address the major underlying

challenges in processing of 2DE gel images e.g., presence of multiple noise, horizontal and

vertical streaking, spot segmentation, spot intensity saturation, overlapping of spots,

geometrical variations, image registrations etc.

Although plenty of commercial software packages are available for analysing 2DE gel

images, their high cost, unavailability of standard image analysis workflow, not-so promising

results and black box development led many research groups to open-source and freeware to

perform analysis of 2DE gel images.

It’s unfortunate that the freeware and open-source resources available do not offers all the

steps required to complete analysis of 2DE gel images. Hence till date not widely used and

adopted and eventually not proved to be the alternative for costly commercial software.

What is Proteomica?

Proteomica Demystifying Proteins developed by us at Proteomics Laboratory, Indian Institute of

Technology Bombay is also motivated by the factors described in the previous section above.

Proteomica is complete open source and freeware alternative solution to commercial software

which provides handy and cost effective option for research community. Proteomica

incorporates all the required operation for complete analysis of 2DE gel images. Proteomica

implement the image domain based analysis workflow which is powered with established and

widely accepted algorithms [][] for processing gel images in each steps. Development of

Proteomica is inspired by []. Also It also incorporates plot and visualisation tool.

Proteomica is a plugin developed for ImageJ and use complete ImageJ framework. Some of

the ImageJ original codes are also modified for its operations hence distributed as bundled

with ImageJ.

For Proteomica we request you to use the Proteomica bundled ImageJ distribution provided

on plugin download page.

Bhushan N Kharbikar,

Biomedical Engineering,

Indian Institute of Technology Bombay

April 2013

First Time User Experiences (FTUE)

Configuring environment

Please follow all the steps provided at link http://imagej.nih.gov/ij/docs/install/ required for

installation of ImageJ as per your operating system (OS) for installing Proteomica bundled

ImageJ distributions.

Setup the required Java environments by setting environmental variables. For details please

refer http://docs.oracle.com/javase/tutorial/essential/environment/paths.html.

Alternatively download the archive (.zip) from link __________________.

Create directory unzip the distribution.

Proteomica is set to use.

Launching Proteomica

Open the directory created after unzipping the to enter you will get directory

structure as follows

Double click on the highlighted above. It will launch the ImageJ.

From all the available menu option in the IJ menu bar go to plugins

Dropdown menu will pop-up. Proteomica distribution is available here and highlighted a

Click on the Proteomica. It launches Splash welcome screen projecting product and its tagline

along with information for copyright.

The programs then load and display the Proteomica’s principal graphical user interface as

below.

Principal Graphical User Interface

Principal screen is divided in three area Product viz., label text area (marked as 1), Copyright

text area (marked as 3) and command area (marked as 2). The command area is further

divided subarea depending group of operations involved in workflow. Also the arrangement

flows as per flow of steps in the image processing workflow for 2DE gel image analysis.

1

2

3

Know the controls

Proteomica do the 2DE image analysis in image domain.

Image domain based approach

In image domain based approach the image alignment step precedes the spot detection.

Reference image has been selected which has maximum no of spots patterns. Other gels

images in match set then aligned to the referenced gel by warping. If alignment is not

satisfactory, the process is then manually intervened and representative spot are selected over

the gels which can be acts landmark points. Algorithm, then realign the images with the

landmark in position and generated smooth interpolated warping for global alignment and

with estimation of intermediary interpolation local alignment is achieved. Effective image

also called fusion image or mean image is created by combining all the aligned images.

Fusion image thus used for further analysis helps in reducing noise and to overcome variable

features and artefacts. The fusion image is then subjected to background subtraction and

intensity normalization. Use of fusion image for spot segmentation and quantification tends

to increase sensitivity over single spots detection. Spot parameters are computed for spots on

individual gel images. The resultant match sets have no missing spots and fidelity of analysis

is high as compared with conventional method. Also the correctness of algorithm is limited

by degree of correctness of gel alignments.

Describing Proteomica GUI

Workflow

Gel analysis starts with setting up an experiment by creating match set

After match-set creation, all the gels added to the match-set are pre-processed to remove all

the noise and background. Pre-procesing can be done repetitively till we get satisfactory

results.

After pre-processing, we have to determine best gel with minimum geometrical distortion and

align all the gels with best one selected as target gel image.

Match and alignment removes all the geometric distortions present in the gel images.

Followed by the same Global gel image is created which would be the combination of all the

gel images present in the match-set of an experiment for analysis of 2DE gel images.

Workflow further advances with the spot detections on the Global gel and after generating the

spot list the spot’s volume are quantified on each individual gels.

Further, the results are generated with spot quantified on each and every gel image signifying

their differential expressions if any. Result also includes few basics statistical parameters

about detected protein’s spots on the analysed 2DE gel images.

Command Buttons

Several command buttons on panel marked as 2 above performs all the important functions in

the image processing and analysis of 2DE gel images. Command buttons controls the

calculations or help in accessing the other functions, as listed below.

Match set button initiate the process and creates the match set

by including gel images in order to setup an experiment

Pre-process button fires the de-noising and background

subtraction filters in order to clean the images and make it ready for the next step in

workflow

Match and Align button pops up the window in which you can

select source and target gel images you want to align along with other options to set up or

refines parameters and perform the matching and alignments and ask you to save the aligned

image.

Global gel button close all the open gel windows and launch

the file selection popups to select all the aligned gel images to create global gel which is the

combination of all the 2DE gel images which is pre-processed and aligned in the previous

steps. Also it generates the stack of all the gel images.

Segment button executes the mark and segment the protein

spots on the Global gel image obtained in previous steps and add the list of detected protein

spots in the spot manger / roi manager.

Quantify button distributed all the protein spots detected in

previous steps to all the gel image in the stack and quantify the corresponding volume of

proteins for the same spot on every gel images.

Spot’s Manager / ROI’s manager button launches the spot /

roi manages which displays and manages all the detected spots on the global gel also it has

capability to save / open (load) / select / add and delete spots from the global gel which are

auto-detected during segmentation of global gel image.

Statistics button generate results for the entire analysis and

also basic statistics describing all the protein spots detected on all the 2DE gel images

included in the match-set for the experiment done as specified

.

3D view button opens the JAVA3D interface to visualize the

3D surface profile of protein spots in gel images.

Profile Plots button plots the 2D pots of the protein spot

intensities. Also it can be operated at real-time or in the live mode. It also saves spot locations

and the plot in the file.

Help button displays the general FAQs about the Proteomica,

Credits and Citations / References used for developing the current version of the Proteomica.

Exit button closes all the windows and exit the Proteomica.

Hovering of the move pointer over the button provides you with the brief information about

function of the said buttons.

Sample gel analysis using Proteomica

Sample gels used in this demonstration are obtained from Proteomics Laboratory,

Department of Biosciences and Bioengineering, IIT Bombay.

Set up an experiment

Create matchset

We start with creating the match set by clicking on Matchset button and selecting all the gels

which we intend to include in the experiments

After selecting all the images click open in file chooser dialog, it will create your matchset by

opening all the selected images

Pre-processing of images

We now proceeds towards the pre-processing steps

Activate the image window frame by clicking on them and then click on pre-processing

button on the Proteomica

Input dialog window pops up asking for the various parameters to be put into it. The filters

are already optimised for best performance based on the default values which appeared in the

popup.

If you still fill to provide different values kindly fill in the same for improving the output of

the step.

Add the end of pre-processing the image is free from most of the noise and also the uneven

background is subtracted from the image under processing.

This step has been repeated for every image added to the image set if you feels that image

requires pre-processing.

This figure below shows the output images before and after pre-processing step.

Gel Image Matching

Match and Align

Next step come is gel image matching. Click on the Match and align button.

Popup dialog comes up asking for the source and target gel image to match. Select target

image as which is best and which may be a reference image and select source image as the

image which you want to align with reference image. And hit done.

Image alignment is started and you see real animated output when registration is in process.

After alignment you can see the image stack of which first image is output aligned image

second image is the target (reference) image and other two in the stacks are distortion vectors

and warping mask.

After successful alignment popup appears asking for the name of the images to save. It will

only save the first image from the stack which the output aligned image in the match set

directory.

Popup message appears displaying the successful alignment of source image with the target /

reference image

Global Gel

Now we move towards creating the Global gel which is the effective combination of the all

the aligned gel images.

Click on Global gel button, file chooser window pops up.

Select only the aligned registered/aligned .jpeg file and click open button on UI.

Popup appears in case if the all gel images are not of the same size. Select copy centre

method from the method dropdown as it provide best results You select other methods to as

which can be decided based on 2DE gel image. Click on OK button.

The process generates the effective Global gel as shown below.

Spot Detection

Segment

Now we have to detect spot on the Global gel. Hit segment button in the spot detection sub

panel.

Window pops up asking for the parameters. Provide necessary parameters and click on start

watershed button.

It initiates watershed segmentation on Global gel image.

Output of the watershed steps appears as follows.

After the successful watershed click on segment button.

Clicking on the segment button opens up the advanced popup asking for the parameters.

Default parameters which appear are already optimised for good performance. Still feel free

to play around with these parameters.

Click OK on the pop-up. It starts detecting the spots and add the detected protein spots on the

Global gel to the ROI/ Spot Manager.

All the detected spots are appeared to be labelled on Global gel and added to ROI/ Spot

Manager.

Before clicking on the start watershed button or segment button, makes sure that Global gel

window is active.

You are able to save the detected spot list in the match-set directory by clicking on the save

button in the ROI/Spot Manager.

Quantify

After detection of the spots on the global gel we have to determine the volume of the protein

spots on the individual gel images.

Quantify does the same for us. Activate the image stack window by click the image stack

window and click on the Quantify button

It put the same spot pattern detected on the Global gel and quantify the volume of the protein

on the individual gel images.

Result and Utility Tools

Statistics

Finally to view results click on statistics button.

Popup message appears stating that generating statistics is under process.

Again popup appears asking how to arrange the results in the result tables. Provide suitable

option and click ok.

Result table starts appearing

Result tables displaying the results which include gel label, spot area, standard deviation,

intDen (Spot Volume), median, kurtosis and skewness.

We can save results file save as and then by giving std extensions (.txt or .csv or .xls)

We can even compute few more results and advanced parameters and display it in result table

to configure the same go to the result window. Click on Results Set Measurements

check all the required parameters and click ok.

3D view visualisation tool

Click on the 3D view button. It launch Java3D viewer in which complete gel and/or even the

part of the gel or protein spot are visualised in 3 Dimensions. For details please follow the

below snapshots

Profile Plot

It helps us to see the 2 dimensional intensity profile of proteins spot on the gel image. Also

the profile of only spots on detected on the stack of the gel image can be visualised.

For visualising the profile of spots detected on the stack of the gel image. The spots must be

loaded in the spot manager / roi manager.

After loading the save spot list in the Spot Manager / ROI manager use ImageJ selection tool

to select the area of the gel for which profile needs to be visualized.

Below the profile plot of spot intensity are displayed.

Interface provides us with option to save the profile plots for 2DE gel images.

We have option to see the live plotting by changing the selection and plot get updated for the

same. This is achieved by clicking on the Live button on the UI.

Help

Help button displays the general FAQs about the Proteomica, Credits and Citations /

References used for developing the current version of the Proteomica.

Exit

Exit button closes all the windows and exit the Proteomica.

References Will be updated soon possible.