What Is a DIA-NN Library-Free Search in a Data-Independent Acquisition (DIA) Proteomics Workflow on ZenoTOF 7600 System?

For research use only. Not for use in diagnostic procedures.

Answer

Label-free quantification relies on comparison of peptides and proteins in their natural (intact) state in consecutive experiments. A label-free approach has been shown to give the largest dynamic range and the highest proteome coverage for identification, but with lower quantification accuracy and reproducibility compared to labeling-based strategies.

In principle, data-independent acquisition (DIA, also called SWATH) approaches allow for the identification of all peptides in a given sample without labeling. However, in contrast to classical data-dependent acquisition (DDA) approaches, the information between the precursor ion and its fragments is lost, resulting in the need for alternative data analysis approaches as well as complex algorithms. The most common approach for analyzing DIA data is a spectral library-based approach in which DDA measurements serve as template for the identification of acquired spectra.

But in addition, a more recently developed workflow – one that does not rely on actual spectral library acquisition – is the library-free approach, where a theoretical spectral library is generated using a FASTA file. The DIA data is then searched against this theoretical library, and the workflow is called "library-free" because a spectral ion library did not have to be acquired separately, an alternative approach currently developed within many different software options (e.g., DIA-NN, Spectronaut, and PEAKS).

In the links below, the DIA-NN library-free workflow for the ZenoTOF 7600 system is described. First, a FASTA file needs to be converted to a theoretical spectral ion library file: https://community.sciex.com/2022/04/29/creating-a-library-from-a-fasta-file-for-library-free-data-analysis/. Next, after the library is generated, the DIA data can be processed with the DIA-ANN software: https://community.sciex.com/2022/04/29/processing-zenotof-7600-system-data-with-dia-nn-software/