Quantifying phosphorylation dynamics in primary neuronal cultures using LC-MS/MS

Cellular processes require tight and coordinated control of protein abundance, localization, and activity. One of the core mechanisms to achieve specific regulation of proteins is protein phosphorylation. Here we present a workflow to monitor protein abundance and phosphorylation in primary cultured neurons using liquid chromatography-coupled mass spectrometry. Our protocol provides a detailed guide on all steps for detection and label-free-quantification of phosphorylated and unmodified proteins of primary cortical neurons, including primary cell culture, phosphoproteomic sample preparation and data-processing, and evaluation. For complete details on the use and execution of this protocol, please refer to Desch et al. (2021).

Automated summary

Cellular processes require precise control of protein abundance, localization, and activity. Protein phosphorylation is a core mechanism for specific regulation of proteins. This study presents a workflow using liquid chromatography-coupled mass spectrometry to monitor protein abundance and phosphorylation in primary cultured neurons. The protocol provides a comprehensive guide for detection and label-free quantification of phosphorylated and unmodified proteins, including primary cell culture, phosphoproteomic sample preparation, data processing, and evaluation.