While generating proteomic spectral libraries today is relatively easy, sometimes a library is needed and there is not time or need to generate a deep library using the traditional data-dependent acquisition approach. Here, a strategy will be outlined to generate a data-independent acquisition (DIA) spectral library from a FASTA file using DIA-NN software for use with a SCIEX QTOF system.  For the most up-to-date information on the use of DIA-NN software and more detailed instructions, please see the DIA-NN software documentation. Download the latest version of DIA-NN software from GitHub.

The first step is to obtain a FASTA file for your organism of interest, from a source such as the UniProt website, shown below. On this website, you can filter by organism to download a FASTA file of interest in either the canonical or the canonical+isoform format.

Then, the DIA-NN software is used to build a spectral library from the FASTA file. No datafiles are needed for this step, as the software will generate a library in silico for the proteins and peptides included in the FASTA file. More details for creating libraries from FASTA files can be found in the library-free instructions in the DIA-NN software documentation.

The dialogue box, below, shows recommended settings for building a library in the DIA-NN software.

• Browse for your FASTA file using the Add FASTA button
• Enter the name for your final library using the Output Library button
• Select FASTA digest for library-free search / library generation
• Select Deep learning-based spectra, RTs and IMs prediction
• Select which modifications are important to your analysis, using the check boxes under Precursor ion generation
• Press Run when you are ready to create the library

It could take up to 1–2 hours to generate the library, depending on its size.

Once created, you can use the generated *.speclib file as you would use any other library to process your SWATH DIA data with DIA-NN.

RUO-MKT-18-14611-A