directDIA is a library‑free, spectrum‑centric, data independent acquisition (DIA) workflow developed at Biognosys. With directDIA, you are able to analyze your DIA data without the need of DDA runs for library generation. This workflow represents the least resource‑intensive and most straight forward DIA analyses approach.

directDIA is possible thanks to the integration of Pulsar into Spectronaut™. Pulsar is Biognosys' proprietary search engine, able to process the highly convoluted DIA data and search it against a protein database (FASTA file). As a result, directDIA allows you to perform protein identification using exclusively your quantitative DIA dataset.

 

Running directDIA in Spectronaut

directDIA is only possible with Spectronaut. During the setting of a directDIA analysis, you will be asked to select a protein database (FASTA file) to perform the search. If you still don’t have your organism database imported into Spectronaut, this is the first thing you must do. To import your FASTA file into Spectronaut, go to the Database Perspective and to the Protein Databases tab (figure 1). Click on import in the bottom‑left corner and browse for your database (FASTA) file. Here, you will have to define the parsing rule for your database so Spectronaut can properly read the information contained in the file. The UniProt parsing rule is already included in Spectronaut. Once you set a parsing rule, you will be able to see a preview of how Spectronaut reads your file. By clicking import in the bottom‑right corner, your database will be available in Spectronaut for further processes.

 

Figure 1. Importing a protein database into Spectronaut before starting a directDIA analysis. Spectronaut will use it later to do the search using Pulsar

 

To perform a directDIA analysis, go to the Review Perspective, click on Load directDIA Experiment in the upper left corner (figure 2). A file explorer will open to let you browse and load your DIA files. Then, a wizard will pop up to guide you through the setup. Add or remove raw files and click next (figure 2). Choose your protein database (FASTA) and click next (figure 3a). Choose your search and analysis settings (figure 3b). Provide information about your experimental setup. You can do this manually or by importing a .xls file with the information (figure 3c). Optionally, you can also provide gene annotation information to get some biological insight about your experiment (figure 3d). In this case, like for the protein database, you need to import the Gene Ontology (GO) annotation file before starting. Finally, by clicking finish, your directDIA analysis will begin.

 

Figure 2. Starting a directDIA analysis. Browse and upload your DIA files. Follow the wizard to complete the process

 

 

Figure 3. Step by step setup of your directDIA analysis: a) choose the database for searching; b) specify your settings for the search, the analysis and the post-analysis; c) configure your experimental conditions; d) optionally, choose a GO annotation file if you want to perform term enrichment analysis

 

To learn more about directDIA visit out ScienceHub here to see some of its applications.

 

To learn more about Pulsar, click here. To learn how DIA is outperforming shotgun proteomics, click here.

 

 

Related content:

Which are the Biognosys solutions for Next Generation Proteomics?

What is Pulsar?

How does DIA differ from classic shotgun proteomics and DDA?

 

 

 

 

Created by SEZ. Last update 2018-03-26 by SEZ