Using XlinkCyNET to visualize protein cross-links in Cytoscape software

Diogo Borges Lima (  diogobor@gmail.com ) Department of Chemical Biology, Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP),13125, Berlin, Germany https://orcid.org/0000-0001-6056-0825 Ying Zhu Department of Chemical Biology, Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP),13125, Berlin, Germany Fan Liu (  iu@fmp-berlin.de ) Department of Chemical Biology, Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP),13125, Berlin, Germany


Introduction
In systems biology, it is essential to understand what biomolecules are involved in different biological processes and how they coordinate to precisely execute an immensely broad range of cellular functions through interactions [1]. An indispensable approach to visualize and interpret these interactions is to use network diagrams. So far, several software tools have been developed for this purpose, such as Biogrid [2] and Cytoscape [3]. Among various options, Cytoscape is arguably one of the most popular network visualization tools. In particular, it offers a great selection of plugins capable of customizing layouts to facilitate the interpretation of the data (e.g., Cerebral [4] and stringApp [5]). However, a plugin that allows the detailed visualization of residue-to-residue connections from cross-linking mass spectrometry (XL-MS) data and annotation of protein domains is still lacking. In light of the suitability and versatility provided by the Cytoscape platform, here we introduce XlinkCyNET, a Cytoscape app that supports the visualization of XL-MS data. We present a step-by-step protocol onto illustrate the usage of XlinkCyNET and describe the functionality functions of important parameters.

Work ow
The following work ow demonstrates how to use XlinkCyNET in Cytoscape.
2.1. In Cytoscape, load a protein interaction network by clicking on File menu -> Import -> Network from le.
2.1.1. The input le must have the following columns, which should be assigned to the correct column

2.2.
Once an interaction network is loaded, XlinkCyNET can be applied automatically to display the residue interaction layout. Two options are currently available: applying the layout to a single node or a set of nodes.
2.2.1. Apply the layout to a single node. After selecting a node, the layout can be applied by clicking on Apps menu -> XlinkCyNET -> Apply layout (or by pressing CTRL + E). The node will be expanded based on the length of the protein (i.e., number of residues). All cross-links will be displayed * as well as annotated protein domains ** ( Figure 2). Note that at least one node needs to be selected, otherwise a warning message will be displayed. To restore the original layout, press CTRL + E again.   Apply the layout to a set of nodes: If more than one node has been selected, the layout can be automatically applied by pressing CTRL + E (Figure 2). The original layout can be restored to all nodes by pressing CTRL + E again.    [7] or Pfam [8] databases by clicking on the respective button. All domain annotations will be automatically incorporated in the table under column "Domain".
2.3.1.5. An input le with node names and domains can be imported through File menu -> Import le.
2.3.1.5.1. Two formats of the input le are currently supported: comma-separated (CSV) and tabdelimited (tab) formats. The CSV les must align with the format in the table (Node Name, Domains **** ). XlinkCyNET can also recognize tab les that are directly downloaded from Uniprot. However, they must contain the following columns: Gene names (in Names & Taxonomy category); and Topological domain (in Subcellular location category).
2.3.1.6. To load protein domains, click on OK button. A message will be displayed con rming the process.
2.3.1.6.1. Once the protein domain annotations have been loaded, they will be inserted into the Cytoscape Node Table in the column domain_annotation. 2.3.1.7. The process can be aborted by clicking on Cancel button.

2.3.2.
To export the protein domains, click on Apps menu -> XlinkCyNET -> Protein Domains -> Export (or by pressing CTRL + D). A message will be displayed showing the selected network and a new window will be opened to set the le name. All protein domains will be exported to a CSV le.

2.3.3.
To customize the colors of protein domains, click on Apps menu -> XlinkCyNET -> Protein Domains -> Set domains color, or press CTRL + T. A new window will be opened, and all loaded domains will be displayed in the table (Figure 5).     2.3.1.5.2. Troubleshooting: If there is a typo in the Domain column, information will not be loaded, and a warning message will be shown in Cytoscape Task History.
2.3.1.6. Troubleshooting: If the domains are being searched in the Supfam/Pfam database and the process has not been completed, a warning message will be displayed asking for con rmation.
2.3.1.7. Troubleshooting: If the domains are being searched in the Supfam/Pfam database and the process has not been completed, a warning message will be displayed asking for con rmation.