Visualisation tools for dependent peptide searches to support the exploration of in vitro protein modifications

Abstract

Dependent peptide searching is a method for discovering covalently-modified peptides-and therefore proteins-in mass-spectrometry-based proteomics experiments. Being more permissive than standard search methods, it has the potential to discover novel modifications (e.g., post-translational modifications occurring in vivo, or modifications introduced in vitro). However, few studies have explored dependent peptide search results in an untargeted way. In the present study, we sought to evaluate dependent peptide searching as a means of characterising proteins that have been modified in vitro. We generated a model data set by analysing N-ethylmaleimide-treated bovine serum albumin, and performed dependent peptide searches using the popular MaxQuant software. To facilitate interpretation of the search results (hundreds of dependent peptides), we developed a series of visualisation tools (R scripts). We used the tools to assess the diversity of putative modifications in the albumin, and to pinpoint hypothesised modifications. We went on to explore the tools' generality via analyses of public data from studies of rat and human proteomes. Of 19 expected sites of modification (one in rat cofilin-1 and 18 across six different human plasma proteins), eight were found and correctly localised. Apparently, some sites went undetected because chemical enrichment had depleted necessary analytes (potential 'base' peptides). Our results demonstrate (i) the ability of the tools to provide accurate and informative visualisations, and (ii) the usefulness of dependent peptide searching for characterising in vitro protein modifications. Our model data are available via PRIDE/ProteomeXchange (accession number PXD013040).

ProteoSushi: A Software Tool to Biologically Annotate and Quantify Modification-Specific, Peptide-Centric Proteomics Data Sets

“…An array of software tools exists for peptide- and PTM-centric analysis including databases, − PTM site assignment, − and visualization. , However, no software tool can quantify and annotate any site and type of modified residues in proteomic data sets. Software tools focused on downstream processing of proteomic data include Perseus, an integrated tool for statistics and comparisons across samples .…”

Peptimetric: Quantifying and Visualizing Differences in Peptidomic Data

“…The nature of peptidomic data makes it highly compatible with computational algorithms and there are already many software programs such as MsViz, PepServe and CLaSS which utilize this fact to map post translational identifications, screen for antimicrobial peptides, and map the feature space of peptidomic datasets respectively [ Martin-Campos et al (2017) ; Das et al (2021) ; Alexandridou et al (2011) ]. In addition to these softwares there are several packages, mostly for R, with similar aims, such as seqinR and Pviz [ Preston et al (2020) ; Sauteraud et al (2021) ]. These tools have their purposes, however, to our knowledge, close to no programs exists which aim to visualize the peptide coverage of complete peptidomes interactively and with easy access.…”

ProteoSushi: a software tool to biologically annotate and quantify modification-specific, peptide-centric proteomics datasets

“…An array of software tools exists for peptide-and PTM-centric analysis including databases [13][14][15][16][17][18] , PTM site assignment [19][20][21][22] , and visualization 23,24 . However, no software tool is able to quantify and annotate any site and type of modified residue(s) in proteomic datasets.…”