Data Independent Acquisition analysis in ProHits 4.0

Abstract

Affinity purification coupled with mass spectrometry (AP-MS) is a powerful technique for the identification and quantification of physical interactions. AP-MS requires careful experimental design, appropriate control selection and quantitative workflows to successfully identify bona fide interactors amongst a large background of contaminants. We previously introduced ProHits, a Laboratory Information Management System for interaction proteomics, which tracks all samples in a mass spectrometry facility, initiates database searches and provides visualization tools for spectral counting-based AP-MS approaches. More recently, we implemented Significance Analysis of INTeractome (SAINT) within ProHits to provide scoring of interactions based on spectral counts. Here, we provide an update to ProHits to support Data Independent Acquisition (DIA) with identification software (DIA-Umpire and MSPLIT-DIA), quantification tools (through DIA-Umpire, or externally via targeted extraction), and assessment of quantitative enrichment (through mapDIA) and scoring of interactions (through SAINT-intensity). With additional improvements, notably support of the iProphet pipeline, facilitated deposition into ProteomeXchange repositories and enhanced export and viewing functions, ProHits 4.0 offers a comprehensive suite of tools to facilitate affinity proteomics studies.

Significance: It remains challenging to score, annotate and analyze proteomics data in a transparent manner. ProHits was previously introduced as a LIMS to enable storing, tracking and analysis of standard AP-MS data. In this revised version, we expand ProHits to include integration with a number of identification and quantification tools based on Data-Independent Acquisition (DIA). ProHits 4.0 also facilitates data deposition into public repositories, and the transfer of data to new visualization tools.

Keywords: Affinity purification coupled to mass spectrometry; Data Independent Acquisition; Laboratory Information Management System; Mass spectrometry; Protein-protein interactions; Proteomics.

Figure 1. ProHits organization and protein interaction scoring

A) The ProHits system consists of two principal modules, a Data Management module and an Analyst module. All mass spectrometers in a facility can be connected to ProHits: scheduled backups of the mass spectrometry data are performed followed by file conversion and database searches. DIA identification is supported by DIA-Umpire and the spectral matching tool MSPLIT-DIA. Peptide and protein identification results are parsed to a Sample defined in the Analyst module. The Samples are defined in a Project → Bait → Experiment → Sample hierarchy. Permissions for different projects are assigned to users in an Admin section. B) Schematic workflow for protein interaction analysis using SAINT through ProHits. Within a project, a user defines which samples should be analyzed and specifies which of those are the controls. The SAINT version (SAINTexpress or standard SAINT) is selected, alongside optional parameters and sample compression level. SAINT uses the quantitative matrix to derive the probability of interactions. Post analysis with SAINT, the data can be visualized or deposited in repositories from ProHits itself.

Figure 2. Handling DIA data in ProHits

A) MSPLIT-DIA workflow in ProHits. In the Data Management module (green boxes) a spectral library can be built from parallel DDA runs (in the current release, only MSGFDB is supported for library generation); all previous analyses within a facility are assembled into an “Archive”. Public repositories (e.g. SWATHAtlas) are also enabled. ProHits uses the spectrum with the highest MSGFDB score as the library spectrum for peptide identification. Identified peptides can be used to generate an assay library to be fed into targeted extraction tools outside of ProHits; alternatively, ProHits has implemented a simple inference mapping of unique peptides to “genes” (here, identified as prot*) for spectral counting and SAINT scoring in the ProHits Analyst module (yellow boxes). B) DIA-Umpire workflow in ProHits. Untargeted identification is performed in the Data Management module, while semi-targeted re-extraction is performed in the Analyst module. Note that SAINT and SAINTexpress intensity models can be used to analyze DIA-Umpire results; alternatively, the results can be analyzed by mapDIA. C) mapDIA workflow in ProHits. The current version has fully implemented the statistical tool mapDIA as a downstream analysis tool post quantification by Umpire Quant; mapDIA also accepts data extracted by targeted extraction tools. mapDIA analysis parameters and experimental design options are selected in ProHits. Visualization of the mapDIA results (here, an example of a time course is shown) can be performed outside of ProHits on the ProHits-viz server (grey boxes).