The sidebar organization for the Preferences and Length is identical, and therefore it is described once for both types of visualization.
The top box, labeled Proteins, lists all identified proteins. Sorting is primarily by the number of identified peptides in descending order and can be switched to alphabetical via “a→z” and “9→0” above. In the Filter field, user can search for specific protein by name defined in database.
Another field, labeled Conditions, lists individual experimental conditions. The name is automatically derived from the filename, as detailed under section Input data. Number in brackets shows the average number of peptides identified for the listed protein in all replicate analyses, which are listed further below, in the field Analyses.
The field named Analyses shows the individual files/analyses belonging to the condition selected in the box above. The analysis name is a truncated file name plus the accession code of the protein selected in the field Proteins. In this field, analyses can be removed (for the current protein or all proteins in the list) or temporarily disabled. This action hides the analysis from the current protein processing but is restored once you select another protein for processing. Such a feature is helpful for outliers. This function is called via right-click within the analyses window.
All the fields, Proteins, Conditions and Analyses allow selection of one/several/all entries. Hence, pooling several/all proteins and/or conditions and/or analyses together is possible. This is done by combining left-click with Shift or Ctrl. Last quarter of this short video shows how users can access cleavage preferences extracted based on one protein or all identified proteins.
Residue distance selects distance from the cleavage (P1/P1’, P2/P2’, … up to P15).
Ruler Reproducibility allows the user to set the reproducibility level and assess data consistency. A 0 value means all peptides in all analyses will be shown, while 100 stands for full reproducibility.
Once the data are ready, their visualization is triggered via the Add data button. This uses the peptides matching the selected proteins, conditions, analyses, and reproducibility filter to plot the cleavage preference bar graph on the right side. Before creating the graph, the program asks for the names of the conditions. By default, it is pre-filled with the distance from the cleavage, the name of the Protein or the number of selected proteins, and the reproducibility level. If the user attempts to add the identical name a second time, the automatic counter turns on (adding 01, 02, 03,…) to the end of the description.
The plot can be exported via the Export plot button and saved as a *.pdf, *.png, or *.svg.
In addition, the data behind the visualization can be exported in text format through the Export data button. This exports the calculated numbers (for plotting in another program). The user can also export the underlying peptides behind the numbers through the Export peptides button.
The Export IceLogo button (under Preferences tab) exports a list of aligned sequences that can be used to visualize protein cleavage motifs using IceLogo. The number of flanking residues upstream and downstream of the cleavage site is set upon clicking the button.
The export takes data selected in the Sidebar and applies the selected filetring for Residue distance and Reproducibility. If multiple conditions are selected, they appear sorted as individual blocks in the exported file and are distinguished by a header.
Using these data, user can go to the IceLogo server (public one or a local one installed on a PC) and plot the IceLogo.
For example - take the data AnPEP digests provided here and its associated database. In DigDig, start the data analysis using *.csv files ProtMix_AnPEP_1-50_1h and ProtMix_AnPEP_1-50_ON. Go to the Preferences, select BSA, 14-3-3, bCA2, Mb and CytC, select both conditions, all analyses, keep residue distance 1 and reproducibility set to 100. In the example shown here, the preferences for both conditions are also drawn for better illustration but plotting them is not necessary for IceLogo data export.
Then press Export IceLogo and the dialogue window for setting the number of flanking residues appears. Set the number to 10.
Now set the file name, e.g.“icelogo.txt” and Save.
The resulting file looks as shown below.
Take the first block of sequences and paste them to the Experimental set window, set Start position to -9.
The resulting IceLogo visualizations is shown below.
If you take the second data block, corresponding to the overnight digestion ProtMix_AnPEP_1-50_ON, the IceLogo looks as below.
The Export Lengths button (under Length tab) exports a number of peptides found for a certain length in each condition used for visualization.