Using ProHits to store, annotate, and analyze affinity purification-mass spectrometry (AP-MS) data

Abstract

Affinity purification coupled with mass spectrometry (AP-MS) is a robust technique used to identify protein-protein interactions. With recent improvements in sample preparation, and dramatic advances in MS instrumentation speed and sensitivity, this technique is becoming more widely used throughout the scientific community. To meet the needs of research groups both large and small, we have developed software solutions for tracking, scoring and analyzing AP-MS data. Here, we provide details for the installation and utilization of ProHits, a Laboratory Information Management System designed specifically for AP-MS interaction proteomics. This protocol explains: (i) how to install the complete ProHits system, including modules for the management of mass spectrometry files and the analysis of interaction data, and (ii) alternative options for the use of pre-existing search results in simpler versions of ProHits, including a virtual machine implementation of our ProHits Lite software. We also describe how to use the main features of the software to analyze AP-MS data.

Figure 1

Suggested modular architecture for the complete ProHits system.

Figure 2

Simplest use of the ProHits VM system. A) The ProHits Lite VM appliance is opened via VirtualBox. B) Screenshot of ProHits VM running on Linux; web browser page (Mozilla Firefox) is open. Note the IP address. C) Standard web browser on the host computer (here, Mozilla Firefox on Mac OS X). ProHits Lite VM may be accessed by typing the IP address from panel B into the host computer browser. Other computers on the network can access the VM via the same IP address. Note that the IP address is dynamically generated and only valid for the current session. The VM window should not be closed while ProHits is in use.

Figure 3

Creating Projects and Users in the “Admin Office” module. A) The ProHits administrator can create new Projects in the Project Manager and new Users in the User Manager. On the right is the view of existing Projects; new Projects can be added by selecting the [Add New] tab. B) Navigation window for creating a new Project. Projects can be associated with current or new laboratories. C) The User manager navigation window. New Users can be added, and associated with different levels of privileges. The privileges of existing Users can also be modified on this page.

Figure 4

Structure of the two main ProHits modules: the “Data Management” (left) and the “Analyst” module (right).

Figure 5

Storage tab of the “Data Management” module. A) Data are organized first by mass spectrometer, then by folders and subfolders. Scheduled file backup is set by the administrator; external raw files can be uploaded to a specific instrument/ folder. B) Raw files can be downloaded, converted, and linked to samples defined in the Analyst module.

Figure 6

Starting a new search tasks in the Data Management module of ProHits. Screenshot of the “Auto Search” tab.

Figure 7

Selecting parameters and samples for “Search Task”. Screenshot from a completed search task after selected parameters are locked in.

Figure 8

Search results page. Chain link icons reflect association to a “Sample” defined in the Analyst module. Search results can be transferred to the Analyst module by selecting “Parse Hits to ProHits Analyst database” and selecting parameters and files when prompted.

Figure 9

Creating Baits, Experiments and Samples. A) In the ProHits Analyst module, select a Project, and then select “Add Gel-free Sample” under the “Create New Entry” category. This will prompt you to create an entry from an existing or new Bait. Selecting “new bait” will open a new dialogue box (shown here). Minimally, you must specify the bait Gene Symbol and Species; pressing the “Get Protein Info” button will prompt ProHits to retrieve the amino acid sequence from its internal database (right inset). B) Once a “Bait” is created, “Experiments” can be added. Protocols can be associated with each Experiment, in addition to controlled vocabularies and free-text annotation. C) Samples can be added and annotated. Multiple Samples may be associated with the same Experiment, and multiple Experiments may be associated with the same Bait.

Figure 10

Uploading search results. A) Once a Bait, Experiment and Sample have been created, the “Upload Search Results” function can be used to import search results from the Mascot, X!Tandem or SEQUEST search engines, or from the Trans-Proteomic Pipeline (TPP). First, select the desired Sample and click on the “Upload” button in the “Options” column. This opens a new window where the file type to upload can be specified, upon which a navigation pane opens. Select the file to be uploaded and the desired filters, and press “Submit”. B) Once search results have been uploaded, the information will be recorded on the upload page. Pressing the graph icon in the “Options” column will open the Sample Report page.

Figure 11

Screenshot of the “Report by Sample” entry page in the Analyst module. All Samples analyzed are displayed: clicking on the multicolored bar expands Experimental details and the linked mass spectrometry files.

Figure 12

Screenshot of “Sample Report Hits” page. Unfiltered hits from the search engine Mascot are displayed.

Figure 13

Screenshot of the search results after applying basic filter sets. The overlap of the data with known interactions in the BioGRID database is indicated.

Figure 14

Screenshot of entry page for the Multiple Sample Analysis “Comparison” page of ProHits. Baits, Experiments or Samples can be selected for combined visualization and analysis.

Figure 15

Screenshot of filtered data from the ProHits “Comparison” page. Baits are shown in columns, preys (hits) in rows. The color-coding maps to quantitative parameters (here number of unique peptides). Links to external databases are provided. Shapes in the individual cells indicate interactions reported in the BioGRID database.

Figure 16

Use of Cytoscape Web to visualize interactions detected by AP-MS directly from ProHits VM. A) A Cytoscape Web view of the data (filtered or unfiltered) can be accessed on any of the report pages (individual reports, comparison view or SAINT report) by pressing the “Cytoscape” icon. Quantitative information embedded in the mass spectrometry file is transferred to Cytoscape Web, either as a node or an edge attribute (examples of the transferred information are listed). B) Example of a report page where the Cytoscape Web information can be accessed. C) Default view of the Cytoscape Web network from panel B.

Figure 17

Exporting peptides. A) In the ProHits Peptide Comparison pages (accessed from the protein-level Comparison page), new functions for exporting peptide information are available. These are identical to the protein-level export function with the exception that only a single protein is analyzed. B) Peptide-level information available in the [Export(select)] function of ProHits. Note that this export function is available for all proteins in the datasets when looking at the protein comparison page, or for a selected protein, as shown here. C) Sample Report: Here, only the deamidated peptides from the protein PRMT5 will be exported to a .csv or .tsv file.

Figure 18

Using ProHits to run SAINT: data example. A) ProHits records all SAINT options, and the list of the samples and controls used to run SAINT, for reporting purposes. B) Screenshot of the SAINT Comparison page of ProHits. A number of display options and filters are available. Manually excluded hits are left on the list, but are greyed out. C) Screenshot of the Excel document resulting from the Export(table) option in panel B. Note that all filters used are detailed and that the proteins that were manually removed from the list are clearly listed (by Genbank Identifier) for increased transparency in reporting.

Figure 19

Managing Protocols. Experimental protocols can be added manually, imported from other projects, and exported to text.

Figure 20

The function “Frequencies” enables the creation of user-specified frequency reports, and the export of project-wide or group-wide frequencies. Here the project-wide frequency set for the TPP results is displayed; this list of proteins can be downloaded.