Year: 2025
Type: preprint
Abstract: This protocol describes the sample preparation for phosphopeptide enrichment from lysed HEK293T cells that were transfected with TurboID, TurboID-LRRK1 or TurboID-LRRK2 and treated with MLi-2, IN04 or DMSO. It is meant to accompany the methods section and manuscript of "Probing the proteome-wide interactomes of Leucine-rich Repeat Kinases 1 and 2 and alterations in their phosphorylation activity". For details on culturing and treatment of cells as well as lysis of these cells, please refer to Protocol "TurboID-phospho protocol" (doi: 10.17504/protocols.io.ewov1drpyvr2/v1) where these steps are explained in detail. This protocol is only meant to describe the sample processing from a cell lysate to enriched phosphopeptides ready to be measured by DIA-based mass spectrometry. This protocol starts with the cell lysate obtained in the above mentioned protocol at step 14 and ends with enriched phosphopeptide that can be analysed by mass spectrometry according to steps 45ff of the same protocol. All steps are described per sample.
Author: Jasmin Jansen
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: gold
OpenAlex ID: W4413872210