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ProtPlot – A Tissue Molecular ProtPlot – A Tissue Molecular Anatomy Program Java-based Data Anatomy Program Java-based Data
Mining Tool: Mining Tool: Screen ShotsScreen Shots
**** DRAFT - undergoing revision ****
Peter F. Lemkin, Ph.D.(1), Djamel Medjahed Ph.D.(2) 1NCI-Frederick; 2SAIC-Frederick, MD 21702
Home page: http://tmap.sourceforge.net/
Revised: 08-26-2004 Version 0.40.1-Beta
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AbstractAbstract• ProtPlot is an open-source Java-based data mining bioinformatic tool
for analyzing CGAP- database derived estimated mRNA tissue EST
expression in terms of a set of virtual 2D-gels
• The estimated mRNA expression is mapped to estimated “proteins”
• It is well known, mRNA expression generally does not correlate well with protein expression as seen in 2D-PAGE gels (Ideker et.al., Science 292:929-934, 2001
• ProtPlot lets you look at the data in new ways and may help in thinking about new hypotheses for protein post-modifications or mRNA post-transcription processing.
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Possible Questions Possible Questions
• ProtPlot may help look at aggregates of CGAP data in new ways:
- Which “estimated proteins” are in a particular (pI,Mw) range?
- Which sets of “proteins” are up or down regulated in cancer(s) and
normal(s) or precancer(s)?
- Which sets of “proteins” are entirely missing in one condition vs. the
other?
- Which sets of “proteins” cluster together across different types of
cancers or normals?
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ProtPlotProtPlot
• It was developed initially as Virtual-2D [Proteomics J, in press], and
upcoming paper on TMAP [Proteomics, in press]
• ProtPlot was derived from an open-source microarray data mining
tool MAExplorer (http://maexplorer.sourceforge.net/) by P. Lemkin
• ProtPlot is a Java application and runs on your computer. You
download and install the application and the data.
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Pseudo 2D-Gel Map Expression DataPseudo 2D-Gel Map Expression Data
• Sample mRNA estimated expression data was obtained for a variety
of human tissue and histology types (normal, pre-cancer, cancer)
using the relative hit rates on cDNA clone libraries. Data from
multiple libraries/tissue were merged
• Pseudo-protein data was computed by mapping the UniGene Ids in
the CGAP libraries to SwissProt AC. The (pI, Mw) was computed
using the SwissProt (pI,Mw) server tool
• These data are assembled into ProtPlot data files called .prp files
described on the Web site.
• ProtPlot then generates an interactive pseudo 2D-gel Map (pIe,Mw)
scatterplot that may be used for data mining
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VIRTUAL2D home: http://proteom.ncifcrf.gov/VIRTUAL2D home: http://proteom.ncifcrf.gov/
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TMAP HOME: http://tmap.sourceforge.net/TMAP HOME: http://tmap.sourceforge.net/
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History of ProtPlotHistory of ProtPlot
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Using ProtPlotUsing ProtPlot
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ProtPlot Menus and User ControlsProtPlot Menus and User Controls
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Initial Screen displaying (pI vs Mw) scatterplotInitial Screen displaying (pI vs Mw) scatterplot
Zoomable pI vs Mw scatter plot
Sample selector
Pull-down menus
Checkbox options
Filter status
Current proteindata
Threshold sliders
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Scatterplot pI vs Mw Limit SlidersScatterplot pI vs Mw Limit Sliders
Mw upper limit
Mw lower limit
pI upper limit
pI lower limit
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ProtPlot: Parameter Threshold SlidersProtPlot: Parameter Threshold Sliders
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ProtPlot: Lower Selectors and CheckboxesProtPlot: Lower Selectors and Checkboxes
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ProtPlot Pull-down MenusProtPlot Pull-down Menus
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ProtPlot Data FormatProtPlot Data Format
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Download ProtPlotDownload ProtPlot
Click on programinstallers
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Download ProtPlot InstallerDownload ProtPlot Installer
Click on Downloadbutton
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Installing ProtPlotInstalling ProtPlot
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Installing ProtPlot (continued)Installing ProtPlot (continued)
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Finished Downloading ProtPlotFinished Downloading ProtPlot
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Starting ProtPlot - Click on the Startup Icon or Use Starting ProtPlot - Click on the Startup Icon or Use the Start menuthe Start menu
C) Press Hidebutton to remove
B) Displays the loading status
A) Click on ProtPlot Startup icon
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ProtPlot MenusProtPlot Menus
• File - select samples, save the state and quit
• View - select viewing options
• Genomic-DBs - enable access to popup Web genomic databases
• Filter - select protein data filter options
• Plot - select primary data mining and scatterplot display options
• Cluster - select cluster distance metrics and perform clustering
• Report - generate popup reports
• Help - popup help menu
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File Menu - Selecting Single Samples,File Menu - Selecting Single Samples,the X-set, Y-set or EP-set of Samplesthe X-set, Y-set or EP-set of Samples
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File Menu - Selecting Samples using Choice MenuFile Menu - Selecting Samples using Choice Menu
Pick specific sample or samples
Sample selector(picked X set)
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Selecting Subsets of Samples for ExperimentsSelecting Subsets of Samples for Experiments
• Current Sample - to look at the expression for any individual sample. E.g., prostate_cancer
• Sample X and Sample Y - to look at the ratio of exprX/exprY where the protein for which the ratio is defined has expression in both the X and Y individual samples. E.g., X is prostate cancer and Y is prostate_normal
• X set of samples and Yset of samples - to look at the ratio of Mean-exprX / Mean-exprY where the protein for which the ratio is defined has expression in both the X and Y samples for at least 1 sample in X and at least 1 in Y. E.g., X set is all cancer and Y is all normal
• Expression Profile set of samples - to look at the expression profile (EP plot or EP report) for any protein. The scatter plot shows mean EP expression. E.g., EP is all samples, or EP is all cancer, etc.
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Plot Display Mode RulesPlot Display Mode Rules
All proteins in the Master Protein Index (mPid) are displayed except for the following:
• In single sample or EP expression mode, do not show missing proteins
• In X/Y sample mode, do not show proteins that are missing in X but present in Y or vice versa. However, if the View option to display this missing data is enabled, then show the missing data as gray spots.
• In X-set/Y-set samples mode, do not show proteins unless they meet the sizing criteria N for both X and Y if enable or if using the missing sets > N filter.
• Normally, plot proteins in a (Mw vs. pI) scatterplot
• If in one of the X/Y ratio modes, may plot (X vs. Y) expression scatterplot instead of (Mw vs. pI)
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Selecting the Current SampleSelecting the Current Sample(those with [>S] have more than S proteins/sample)(those with [>S] have more than S proteins/sample)
Pick specific sample
Slider to set S the# proteins/Samplefor the sample to beused
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Report Menu - Listing # Proteins in All SamplesReport Menu - Listing # Proteins in All Samples
Popup report
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Selecting the X SampleSelecting the X Sample
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Selecting the X-set of SamplesSelecting the X-set of Samples
Pick multiple samples
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Selecting the Y SampleSelecting the Y Sample
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Selecting the Y-set of SamplesSelecting the Y-set of Samples
Pick multiple samples
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Selecting the Expression Profile (EP) Set of SamplesSelecting the Expression Profile (EP) Set of Samples
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Listing Sample AssignmentsListing Sample Assignments
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Defining the X and Y Condition Set NamesDefining the X and Y Condition Set Names
A.1 (default X set)
A.2set to ‘cancer’
B.1 (default Y set)
B.2set to ‘normal’
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Click on a Spot to Select the ProteinClick on a Spot to Select the Protein
Report on protein
Protein selected
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Select a Protein by SwissProt ID or ACCSelect a Protein by SwissProt ID or ACC
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File Menu - Save & Restore the Data Mining StateFile Menu - Save & Restore the Data Mining State
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File Menu - Updating the Program and PRP dataFile Menu - Updating the Program and PRP data
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Updating ProtPlot Program from the Proteom ServerUpdating ProtPlot Program from the Proteom Server
Asks you to verify thatyou want to update theprogram
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View Menu - Display OptionsView Menu - Display Options
Modifies how data is displayed.Some of the options are also inthe checkboxes below
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Genomic-DBs MenuGenomic-DBs Menu
Select the database to useif you enable Web GenomicDatabase access
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Bringing up a Genomic Server by Clicking on SpotBringing up a Genomic Server by Clicking on Spotif you Enabled Genomic DB Accessif you Enabled Genomic DB Access
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Filter Menu - Data Filter Options for Filter Menu - Data Filter Options for Single SampleSingle Sample
Data filter which proteins will be visible. The results may be usedin the scatterplot, reports and as theset of proteins used in clustering
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Filter Menu - Data Filter Options for Filter Menu - Data Filter Options for X/Y RatioX/Y Ratio
Data filter which proteins will be visible. The results may be usedin the scatterplot, reports and as theset of proteins used in clustering
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Filter Menu - Data Filter Options for Filter Menu - Data Filter Options for EP-setEP-set
Data filter which proteins will be visible. The results may be usedin the scatterplot, reports and as theset of proteins used in clustering
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Filter Types - AvailableFilter Types - Available
• By Proteins > 200 Kdaltons, Mw and pI within ranges
• By tissue types
• By expression value range
• By expression X/Y ratio range (either inside or outside range)
• By t-Test of X-set and Y-Set samples < p-value threshold
• By min # samples in X &Y or EP sets > N samples threshold
• By missing proteins in X or Y set with other set > N samples threshold
• By number of samples for the protein > N samples threshold or < N samples threshold
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Applying Applying Expression RangeExpression Range Filter [0.455 : 1.0] Filter [0.455 : 1.0]
Lower expressionrange slider
Upper expressionrange slider
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Applying the ‘outside’ Applying the ‘outside’ X/Y Ratio RangeX/Y Ratio Range Filter Filter< 0.1000 OR > 10.000< 0.1000 OR > 10.000
Lower ratio range slider
Upper ratio range slider
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Applying the t-test (p=0.05) Filter X/Y setsApplying the t-test (p=0.05) Filter X/Y setsMin 4 samples for X and Y, S>=2000 proteins/sample Min 4 samples for X and Y, S>=2000 proteins/sample
p-value threshold slider
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Applying the t-test (p=0.05) Filter X/Y setsApplying the t-test (p=0.05) Filter X/Y setsMin 7 samples for X and Y, S>=2000 proteins/sample Min 7 samples for X and Y, S>=2000 proteins/sample
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Saving Filter Set of Proteins - For Future FilteringSaving Filter Set of Proteins - For Future Filtering
Saved FilterResults [F: #]
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Plot Menu - Display Mode and OptionsPlot Menu - Display Mode and Options
Plot modes for single sample, X, Y or EP sets of samples, expression or ratio data
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Plotting Display ModesPlotting Display Modes
• Show Current Sample - to look at the expression for a single sample
• Show Mean Expression-Profile set of samples - to look at the mean expression for a subset of samples
• Show X-Sample /Y-Sample Y - to look at the ratio of two individual samples
• Show X-set samples / Y-set samples - to look at the ratio of Mean-exprX / Mean-exprY for two sets of samples (X and Y sets)
• If in one of the X/Y ratio modes, may plot (X vs Y) expression scatterplot instead of default (Mw vs. pI) scatterplot
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Plot Display Mode - Current SamplePlot Display Mode - Current Sample
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Plot Display Mode - Mean of EP Set of SamplesPlot Display Mode - Mean of EP Set of Samples(N >= 14, S >= 2000)(N >= 14, S >= 2000)
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Plot Display Mode - X Sample (Red) + Y Sample (Green)Plot Display Mode - X Sample (Red) + Y Sample (Green)
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Plot Mode - Sample Xvs Sample Y Plot Mode - Sample Xvs Sample Y Expression ScatterplotExpression Scatterplot
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Plot Mode - X Sample / Y Sample Colormap Plot Mode - X Sample / Y Sample Colormap
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Plot Mode - Sample X vs Sample Y Plot Mode - Sample X vs Sample Y Expression ScatterplotExpression Scatterplot
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Plot Mode - Mean X-set / Mean Y-set SamplesPlot Mode - Mean X-set / Mean Y-set Samples
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Plot Mode - Mean X-set vs Mean Y-set Plot Mode - Mean X-set vs Mean Y-set Expression ScatterplotExpression Scatterplot
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Plot Mode - Showing Proteins With Either X or Y Plot Mode - Showing Proteins With Either X or Y Samples Missing as Gray ‘+’ or Boxes Samples Missing as Gray ‘+’ or Boxes
Missing X or Y proteins legend
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Plot Mode - Popup Expression Profile Plot for 1 Plot Mode - Popup Expression Profile Plot for 1 Protein - Click on a Different Spot to Change the PlotProtein - Click on a Different Spot to Change the Plot
Popup list of samples and their expression for that protein
EP plots withzoom and curve options
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Cluster Menu - Find Proteins with Similar ExpressionCluster Menu - Find Proteins with Similar Expression
Clustering uses thedistance slider to determine which proteins are similar tothe current protein
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Clustering on Selected Protein - Scatterplot with Clustering on Selected Protein - Scatterplot with Cluster Member Proteins Shown with Black BoxesCluster Member Proteins Shown with Black Boxes
Cluster display shows proteins passingcluster test with black boxes. Other proteinsare those that passed the data filter.
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Clustering on Selected Protein (All Samples) D<0.69Clustering on Selected Protein (All Samples) D<0.69
Dynamic cluster report showingthe cluster distance < threshold
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Scrollable EP Plots for Clustered ProteinsScrollable EP Plots for Clustered Proteins
Click on bar to show sample and value
Scroll through all proteins
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Clustering on Selected Protein - Cluster Report with Clustering on Selected Protein - Cluster Report with Silhouette Plot Sorted by Cluster DistanceSilhouette Plot Sorted by Cluster Distance
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Saving Cluster Set of Proteins - For Future FilteringSaving Cluster Set of Proteins - For Future Filtering
Saved ClusterResults [C: #]
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Report Menu - Options are Display Mode DependentReport Menu - Options are Display Mode DependentCurrent Sample ModeCurrent Sample Mode
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Report Menu - Options are Display Mode DependentReport Menu - Options are Display Mode DependentRatio Mode optionsRatio Mode options
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Report Menu - Options are Display Mode DependentReport Menu - Options are Display Mode DependentMean EP Expression ModeMean EP Expression Mode
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Popup Report for the Filter X/Y setsPopup Report for the Filter X/Y setsMinimum S>=3465 Proteins/Sample Minimum S>=3465 Proteins/Sample
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Popup Report for the t-test (p=0.05) Filter X/Y setsPopup Report for the t-test (p=0.05) Filter X/Y setsMin 7 Samples for X and Y, S>=2000 proteins/sample Min 7 Samples for X and Y, S>=2000 proteins/sample
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Popup Report Expression Profile Values for Filtered Popup Report Expression Profile Values for Filtered Proteins (min N>= 14 samples, S >=2000)Proteins (min N>= 14 samples, S >=2000)
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Popup Report of Samples in Expression Profile Set Popup Report of Samples in Expression Profile Set for the Currently Selected Proteinfor the Currently Selected Protein
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Popup Report # of proteins/sample for All SamplesPopup Report # of proteins/sample for All Samples
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Popup Report All X, Y, EP Sets Sample AssignmentsPopup Report All X, Y, EP Sets Sample Assignments
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Help Menu - Popup Web Browser DocumentsHelp Menu - Popup Web Browser Documents
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Saving the Current Data-mining Session StateSaving the Current Data-mining Session State
Save as new startupstate file
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Changing the State to a Previous Data-mining SessionChanging the State to a Previous Data-mining Session
Opening the data-miningstate to a previous session
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Changing the Filter Set of Proteins to a Changing the Filter Set of Proteins to a Previously Saved Filter SetPreviously Saved Filter Set
Changing the Filter set of proteins to previously saved set
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ReferencesReferences
• Medjahed D, Luke BT, Tontesh TS, Smythers GW, Munroe DJ, Lemkin PF, TMAP poster, Swiss Proteomics Meeting, Geneva, Dec, 2002.
• Medjahed D, Smythers GW, Powell DA, Stephens RM, Lemkin PF, Munroe DJ, VIRTUAL2D: A Web-accessible predictive database for proteomics analysis, Proteomics, 2003, (in press, Feb).
• Medjahed D, Luke BT, Tontesh TS, Smythers GW, Munroe DJ, Lemkin PF, "TMAP" (Tissue Molecular Anatomy Project), an expression database for comparative cancer proteomics. Proteomics, 2003, (in press, June).
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ReferencesReferences
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