Abstract
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.
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Martinez-Val, A., Bekker-Jensen, D.B., Hogrebe, A., Olsen, J.V. (2021). Data Processing and Analysis for DIA-Based Phosphoproteomics Using Spectronaut. In: Cecconi, D. (eds) Proteomics Data Analysis. Methods in Molecular Biology, vol 2361. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1641-3_6
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DOI: https://doi.org/10.1007/978-1-0716-1641-3_6
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