Protein Interaction Network Analysis Using Cytoscape and NDEx

In this tutorial, you will:

  • Generate a protein interaction network from tabular interaction data.
  • Extend the network with protein interactions from public resources.
  • Format the network for effective visualization.
  • Open networks from the web in Cytoscape.
  • Save networks to NDEx.

Background

The data used for this protocol is from an affinity purification-mass spectrometry experiment which identified interactions between human and HIV proteins by Jäger et al. In this quantitative AP-MS experiment, a relatively small number of HIV bait proteins are used to pull down a larger set of human prey proteins.

Note: This tutorial does not describe how to pre-process the raw AP-MS data, the data used here is already scored and filtered.

Note: You can also choose to skip the loading of the data to generate an interaction network, instead downloading a previously created network from NDEx.

Setup

This protocol requires:

To update the required Apps:

  • Launch Cytoscape 3.7.2.
  • Use the App Manager to update all Apps to the latest version available.

Option 1:Create the Interaction Network from Tabular Data

  • Load the network via File → Import → Network from File...., and select the Example data.
  • In the Import Network from Table dialog, select the Bait column as Source Node and Prey column as Target Node.
  • Designate the AP-MS Score as an Edge Attribute.
  • For all other data values, designate them as Target Node Attribute, by clicking in the column header and selecting the red spreadsheet icon.
  • Click OK to import the network.

Option 2: Import the Interaction Network from NDEx

  • Search the AP-MS Demo Data network in NDEx by typing "ap-ms demo data" in the NDEx Search Box and clicking the search icon .
  • The cyNDEx Browser window will pop up... Click the Import button next to the AP-MS Demo Data network, then close the pop up window.

The Interaction Network

Cytoscape will now display the interaction network with a default style and a grid layout (When loading from tabular data as described in Option 1, the layout may be different depending on your Cytoscape settings. Don't worry, this will not matter.)

Add Human PPI to the Network

Now extend the network by adding known protein-protein interactions between the human proteins. This interaction data will be obtained via the NDEx Integrated Query (IQuery) using the human prey proteins from our network.

  • Click the Filter tab in the Control Panel
  • Click the "Plus" sign and add a Column Filter
  • Then, use the Dropdown element to select the Uniprot column
  • Now, select all the genes in the name column by selecting the top item, then scroll down and hold down Shift and then selecting the last entry.
  • Click Ctrl+C (Command+C if using a Mac) to copy.

  • Note: there is a minor issue with selection in which all of the columns are highlighted, not just the one that is selected. While confusing, the selection works correctly.

Add Human PPI to the Network

  • Open your browser and go to the NDEx website
  • Paste the list of gene symbols into the iQuery text box (red inlay)
  • Click the search icon to run your query

Add Human PPI to the Network

In the following results screen, select the Interactomes tab to view a list of reference PPI sources available in NDEx. In this tutorial, we will use the HumanNet v3 - FN interactome, but you are welcome to experiment with different sources.

  • Select the HumanNet v3 - FN result. We will use the default query type (Direct) to add direct interactions with our AP-MS genes.
  • Click the search icon to run your query.

Add Human PPI to the Network

The interactome query result is displayed in a new tab within the NDEx webapp as shown below. Here, you can refine your query by changing the query type or save the query result to your NDEx account (must be signed in) for later use. In this example, we will simply import the result in Cytoscape.

  • Click the Open in Cytoscape button at the bottom right of the page.
  • Note: in order for this to work, you must have Cytoscape installed and running on your computer.

Add Human PPI to the Network

The query result is automatically imported in Cytoscape and the resulting network, named Direct query result on network - HumanNet v3 - FN, contains known interactions between human query (prey) proteins and their targets.

Preparing to Merge Networks

Before merging our two networks, we should clear the Column Filterthat was applied previously. To do so:

  • Select again the AP-MS network
  • Click the Filter tab in the Control Panel
  • Click the "X" sign next to the Column Filter to remove it

Merge Networks

Now we are ready to incorprate the human protein interactions from the IQuery result: we are going to merge the AP-MS network with the HumanNet XN query result, matching nodes using the official gene symbols stored in the name column.

  • Go to Tools → Merge → Networks...

  • In the Available Networks list, select both networks and click the right arrow to add them to the list of Networks to Merge

  • Expand the Advanced Options interface

  • In the Matching Columns field, select name for the AP-MS network and name for the IQuery result network if they are not already pre-set

  • Click the Merge button

The Merged Network

When the merged network first loads, it will have the Cytoscape default style (inherited from the AP-MS network) and the Cytoscape default layout (Prefuse Force Directed Layout). All nodes look the same and this makes it difficult to interpret.

Visual Styling Based on Continuous Numeric Data

Let's now improve the visualization of our merged network by creating continuous mappings to style nodes and edges based on the data associated with their properties.
Note: if you are not familiar with Continuous Mapping of data to visual styles in Cytoscape, please review this short (1 min) video before moving forward to the next section.

Visual Styling Based on Continuous Numeric Data

To begin, we will color the nodes based on their Jurkat Score property (associated to all human query nodes); then we will set the width of the edges based on their AP-MS Score property:

  • In the Style tab of the Control Panel, create a new style named AP-MS Jurkat Score.

  • Create a continuous mapping for the node Fill Color using the JurkatScore column, then select a purple gradient from the ColorBrewer palettes.

  • Switch the deafult node Fill Color to yellow to highlight the HIV proteins.

  • Create a passthrough mapping for node Label using the name column.

  • Now switch to the Edge tab and create a continuous mapping for edge Width using the AP-MS Score column and set the width values to a range from 1 to 10.

Visual Styling Based on Discrete Data

Let's now further enhance our network's visualization by applying a discrete mapping to better differentiate its edges.
Note: if you are not familiar with Discrete Mapping of data to visual styles in Cytoscape, please review this short (1 min) video before moving forward to the next section.

Visual Styling Based on Discrete Data

We will now create a discrete mapping and color the edges to distinguish HIV-Human interactions from Human-Human interactions:


  • Create a Discrete mapping for the edge Stroke Color (unselected) property using data from the interaction column

  • Choose a GREEN color for all interactions of type interacts with

  • Then change the default property color to RED

The Improved Visualization of the Network

The visualization of the network now highlights the AP-MS experimental data (GREEN edges), as well as additional known interactions from HumanNet (RED edges), with a PURPLE gradient node color indicating the Jurkat Score for our human query genes.

Inspecting Visualized Interactions

Zooming in on the network, specifically the POL and PR HIV nodes, we can see a set of interactions found in the original paper by Jäger et al, which represent binding of POL and PR with the translational initiation complex EIF3.

Pathway Enrichment with NDEx IQuery

IQuery lets you find pathways relevant to the host (human) proteins. Go back to the IQuery web page and select the Curated Pathways tab to view a list of pathways from several sources.

Pathway Enrichment with NDEx IQuery

There are three ways to sort the enrichment query results, selected by the appropriate radio buttons:

  • By default, pathways are sorted by the Similarity method

  • Use the radio buttons to choose the Overlap or p-Value methods and the order of the results will change accordingly.

  • Click on the blue Info Icon next to the radio buttons to see the documentation for each sorting method.

  • Each pathway result shows its 3 scores and the one currently used for sorting is shown in bold


Open in Cytoscape from IQuery

  • Select the 5th result GO:0006413 (translational initiation), then click the Cytoscape icon to open the network in Cytoscape as you already did earlier on.

  • IQuery has added a column called querynode to nodes in the network to indicate that these are the proteins in the network that match your query. You can use that information to highlight those nodes.

  • Replace the existing Discrete mapping for node Fill Color to instead use the querynode column

  • Choose a BRIGHT GREEN for the case where querynode is true to distinguish the cluster of EIF3 family proteins that are in common between the original AP-MS network (human prey proteins) and this GO:0006413 (translational initiation) pathway.

Save to NDEx from Cytoscape

From Cytoscape, you can easily save the modified pathway network directly to your NDEx account.

  • To do so, select the GO:0006413 (translational initiation) network in the Network Tab of the left hand panel.
  • Click the button in the top tool bar, then choose Export Network to NDEx
  • The NDEx Save Dialog will be displayed, but the Export Network to NDEx Button in the lower right corner will be deactivated (red oval) because Cytoscape is not yet signed in to an NDEx account.
  • Click on the account manager box in the upper right hand corner and choose Add new profile...
  • Enter your NDEx account name and password, then click the Sign In button. The Export Network to NDEx button will now be enabled allowing you to complete the save operation.

Save to NDEx from Cytoscape

  • Now visit the NDEx website at www.ndexbio.org and log in to your account

  • In your account page, you will see the GO:0006413 (translational initiation) network that you just exported from Cytoscape at the top of the list.

  • From your NDEx account page, you can easily:
    • Share networks with colleagues
    • Perform different types of Neighborhood Query
    • Edit network attributes
    • Request a DOI

References