Data Processing and Analysis for DIA-Based Phosphoproteomics Using Spectronaut

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Data-independent acquisition (DIA) for liquid chromatography tandem mass spectrometry (LC-MS/MS) can improve the depth and reproducibility of the acquired proteomics datasets. DIA solves some limitations of the conventional data-dependent acquisition (DDA) strategy, for example, bias in intensity-dependent precursor selection and limited dynamic range. These advantages, together with the recent developments in speed, sensitivity, and resolution in MS technology, position DIA as a great alternative to DDA. Recently, we demonstrated that the benefits of DIA are extendable to phosphoproteomics workflows, enabling increased depth, sensitivity, and reproducibility of our analysis of phosphopeptide-enriched samples. However, computational data analysis of phospho-DIA samples have some specific challenges and requirements to the software and downstream processing workflows. A step-by-step guide to analyze phospho-DIA raw data using either spectral libraries or directDIA in Spectronaut is presented here. Furthermore, a straightforward protocol to perform differential phosphorylation site analysis using the output results from Spectronaut is described.

Original languageEnglish
Title of host publicationProteomics Data Analysis
EditorsDaniela Cecconi
Number of pages13
Publication date2021
ISBN (Print)978-1-0716-1640-6
ISBN (Electronic)978-1-0716-1641-3
Publication statusPublished - 2021
SeriesMethods in Molecular Biology

ID: 280173836