Philosopher: a versatile toolkit for shotgun proteomics data analysis

To the Editor: Here we introduce Philosopher (https://philosopher.nesvilab.org), a free, open-source, versatile, and robust data analysis toolkit designed to bring easy access to a powerful and comprehensive set of computational tools for shotgun proteomics data analysis.

Computational analysis is a central component of any modern experiment, and mass spectrometry-based proteomics is no exception. As technologies continue to rapidly advance with respect to throughput and sensitivity, bioinformatics tools must keep pace with large-scale experiments. While existing proteomics tools, such as the Trans-Proteomic Pipeline (TPP)¹, MaxQuant², and PeptideShaker³ are capable of performing high-quality analyses, all require installation and are dependent upon specific operating systems, libraries, and other software. Managing these tools can be a daunting task, even for research groups with substantial bioinformatics expertise. This is particularly true when experiments demand high performance configurations such as GNU/Linux clusters or cloud computing. To address this challenge, we initially built and deployed Docker containers with different applications for proteomics, which in part inspired the creation of the BioContainers resource for different bioinformatics fields⁴. Though efficient for packing and sharing resources, we found that chaining different applications with custom implementation of established algorithms in a transparent and dependency-free way was still a challenge for containerization. The Philosopher toolkit integrates high-performance algorithms and existing tools (Fig. 1), and is a dependency-free, fast, and comprehensive proteomics pipeline, able to rapidly process even the most complex proteomics data sets with efficient resource management.

Figure 1. Philosopher provides easy access to a complete suite of computational proteomics tools.

Functions include protein sequence database download and formatting, database searching, peptide-spectrum match validation with PeptideProphet, peptide evidence integration with iProphet, post-translational modification (PTM) localization with PTMProphet, and protein inference with ProteinProphet. Philosopher has multiple options for FDR scoring; performs label-free and labeling-based quantification, PTM annotation, generation of multi-level reports, and quantification comparison between samples. MSFragger and TMT-Integrator must be downloaded separately.

Philosopher includes the database search engine Comet and can use the high-performance search engine MSFragger⁵ as a separately-downloaded tool. For downstream processing of peptide-spectrum matches (PSMs), Philosopher includes key components of TPP. In addition, it implements best practices for False Discovery Rate (FDR) filtering and data summarization that are not readily available within the TPP, such as picked FDR, two-dimensional or sequential (at PSM and protein levels) filters, and additional options for dealing with peptides whose sequence is present in multiple proteins (e.g. razor peptide approach). As quantification is frequently the goal of modern proteomics experiments, Philosopher includes algorithms for both label-free quantification and isobaric label-based quantification (TMT or iTRAQ). Precursor spectral intensities are retrieved following a method described previously⁶. Protein-level quantification is estimated using the sum of the three most intense supporting ions. Alternatively, Philosopher can use TMT-Integrator (http://tmt-integrator.nesvilab.org/) as an external tool or output files can be used with downstream quantification and statistical tools such as MSstats⁷. The rich reports generated by Philosopher are also compatible with other software such as PDV for visualization of peptide assignments to MS/MS spectra⁸ and CRAPome/REPRINT (https://reprint-apms.org/) for interactome scoring and network visualization using affinity purification mass spectrometry data.

Philosopher is scalable from laptops and desktops to high-performance servers, and can be incorporated in workflow managers such as Galaxy-P⁹. The power of Philosopher is well illustrated in the recent CPTAC3 consortium publication, where over 800 spectral files from a clear cell Renal Cell Carcinoma cohort, whole proteome and phosphoproteome data, were analyzed in less than 24 hours each with Philosopher and MSFragger on a single Linux server¹⁰. While Philosopher is most flexible as a command line tool, its core functions can easily be accessed through our widely used graphical user interface FragPipe (https://fragpipe.nesvilab.org/). In summary, installation and management of computational proteomics tools can be a daunting task, particularly when workflows need to be repeated on different operating systems or scaled from desktop to servers or cloud systems. Philosopher is a versatile and easy-to-use cross-platform toolkit for streamlined proteomics analysis that does not require installation or configuration and can perform full analyses (from spectral files in open mzML/mzXML format to peptide and protein reports) in a single command.