Pep2Graph: A standalone tool to analyse proteolytic cleavages by proteases from gel‐based mass spectrometry data

Proteases are enzymes that regulate substrates via proteolytic activation and coordinate essential cellular functions including DNA replication, DNA transcription, cell proliferation, differentiation, migration and apoptosis. However, techniques to identify proteolytic events in a high‐throughput manner is limited. PROtein TOpography and Migration Analysis Platform (PROTOMAP) is a technique that relies on mass spectrometry‐based proteomics to globally identify the shifts in the in‐gel migration of proteins and their corresponding fragments that are obtained by proteolysis. However, user‐friendly software tool to analyse the proteomic data to identify proteolytic events is needed. Here, we report Pep2Graph, a user‐friendly standalone tool that integrates peptide sequence information from in‐gel proteomics and presents the data as two‐dimensional peptographs with in‐gel migration, sequence coverage and MS/MS spectra counts. Pep2Graph (http://www.mathivananlab.org/Pep2Graph) allows users to utilize in‐gel proteomics data to study proteolytic events that may play a significant role in normal physiology and pathology.

tify proteolytic events in a high-throughput manner is limited. PROtein TOpography and Migration Analysis Platform (PROTOMAP) is a technique that relies on mass spectrometry-based proteomics to globally identify the shifts in the in-gel migration of proteins and their corresponding fragments that are obtained by proteolysis. However, user-friendly software tool to analyse the proteomic data to identify proteolytic events is needed. Here, we report Pep2Graph, a user-friendly standalone tool that integrates peptide sequence information from in-gel proteomics and presents the data as two-dimensional peptographs with in-gel migration, sequence coverage and MS/MS spectra counts. Pep2Graph (http://www.mathivananlab.org/Pep2Graph) allows users to utilize in-gel proteomics data to study proteolytic events that may play a significant role in normal physiology and pathology.

K E Y W O R D S
peptides, peptograph, proteases, PROTOMAP Proteolytic cleavage or proteolysis is the breakdown of proteins into shorter fragments and is catalysed by enzymes called proteases [1][2][3][4]. Hence, proteases regulate the stability, localization and activity of numerous proteins [5,6]. Proteases are ubiquitous and are involved in multiple biological processes including growth, digestion, wound repair, protein catabolism and homeostasis [7,8]. Additionally, proteases regulate essential cellular functions including proteolytic activation, DNA replication, DNA transcription, cell proliferation, differentiation, migration and apoptosis [9]. Considering the importance of proteases in both health and disease conditions, there has been significant efforts to understand the classes of proteases and the functional activity of the substrate peptides [6,10]. context of cancer, proteases are implicated in invasion and metastasis, which are the major hallmarks of cancer [10]. Notably, proteases play important role in reshaping the tumour microenvironment [11].
Though high throughput mass spectrometry-based proteomics studies are performed routinely to identify protease substrates [12,13], user-friendly software tool to analyse such proteomic data to identify proteolytic events are needed.
Currently, PROtein TOpography and Migration Analysis Platform (PROTOMAP) [12,14], an existing software, is used by various research groups to plot peptographs [12,13,15]. This software scripts were written using perl programming language and has been designed to work only with output from DTASelect, a software package to group

F I G U R E 3
Snapshot of data viewer tab once the input data is loaded into the application. The filter option is available to select the data for a specific gene of interest and the save option to download data as a excel or a csv file on to the local computer.

F I G U R E 4
Pep analysis dashboard with various filters, tables, options to analyse and examine the data. The Plot quick peptograph option can be used to plot a peptograph with filtered data associated with the checkboxes and filters.
F I G U R E 5 Schematic representation of how to plot peptographs. Radio buttons can be used to select genes from input or from custom uploaded txt file. Checkboxes to plot peptograph with selected checkbox associated filtered data. Select colours list box to add colours to each experiment. Various options are available to customise the plot like title, quality, y-axis, etc.
help the users to visualise the peptides and their start and end position. In case of multiple peptides overlapping, the hover functionality will show all the peptides involved as a list. The y-axis is shared between the peptograph plot, spectral count plot with different x-axis and the user has the option to update the y-axis with the defined molecular weights and gel bands. The users have multiple options to edit the fonts, colour, colour intensity and the quality of image prior to saving an image compatible for publications.
Overall, Peptograph plots in-gel migration in the vertical dimension (high-to low-molecular weight, top to bottom) and sequence coverage in the horizontal dimension (N to C terminus, left to right). Peptograph visualises peptides from proteolytic cleavage for different experiment F I G U R E 6 Peptographs plotted from the example dataset for different proteins for four experiments (four different colours representing each).
groups, having assigned a unique colour for each experiment group ( Figure 6). This helps users to analyse and understand the behaviour of peptides under each experiment group. When the user hovers the mouse on to the rectangular patch (peptide), it shows the sequence of the peptide and the start and end position of the peptide and on the right hand. The spectral count plot shows the magnitude of the proteolytic cleavage for that protein at that specific band in that respective experimental group. The pep analysis tab has three data tables visualised as a dashboard filter section on the top and a button to plot a peptograph for the filtered data. This helps users to analyse proteins undergoing various alterations like any changes in the molecular weight of the protein after proteolytic cleavage in each experimental group.