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DIA-NN software is a powerful software tool for processing data independent acquisition (DIA) proteomics datasets. It uses neural networks and other algorithms to identify and quantify peptides and proteins from DIA data and is specifically optimized for fast chromatography. Here, we share some processing settings that we have found work well for processing ZenoTOF 7600 system data.
Processing data using a spectral library built with OneOmics suite
Spectral libraries can be generated in OneOmics suite using the ProteinPilot app. The group file created from the ProteinPilot app can be converted to a DIA-NN software-compatible file by performing a short processing run in the Extractor app, which extracts the library information and writes a *.txt file into your Results folder. It is important to use the Exclude Modifications option in the Extractor app at this step, as the DIA-NN software does not yet recognize all the modifications that the ProteinPilot app can identify using the Thorough search option. Recommended filters are 1% global protein and 1% global peptide. If you have multiple datasets that you want to combine into a single library, you can do so with the Extractor app, which will align the retention times and merge the 2 libraries at the protein level.
There is also a new Ion Library app, that streamlines the creation of the ion library. This app uses ProteinPilot to search a set of DDA files, perform protein grouping, and then output an ion library that is compatible with DIA data processing in both OneOmics suite and DIA-NN software.
Once the ion library has been generated in Extractor or using the Ion Library app, download the ion library from the Data Store. The ion library generated in Extractor will be named, full_ion_library.txt and will be located in the same folder that you selected your results to be written to. The ion library generated using the Ion Library app will be located in the specified results folder and will be a text file for use in DIA-NN. Now, you can process your SWATH DIA data in DIA-NN software using the library you just built with OneOmics suite. Download the latest version of DIA-NN software from GitHub.
The settings shown below can be used as a recommended starting point to process data in DIA-NN software collected using SCIEX QTOF instruments.
Many applications will benefit from the use of the “–library-headers…,” “–report-lib-info” and “–relaxed-prot-inf” commands. Other commands can be added to your processing workflow in the Additional options window, by entering command line instructions.
The DIA-NN software documentation on command line tools provides the following descriptions of the flags recommended, above. Note: each flag must start with a double dash (doesn’t show up well in the text below).
--relaxed-prot-inf
--report-lib-info
--library-headers modification_sequence,prec_z,q1,rt_detected,q3,relative_intensity
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