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

• Published in: STAR protocols Vol. 3; no. 1; p. 101063
• Main Authors: Desch, Kristina, Schuman, Erin M., Langer, Julian D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.03.2022; Elsevier
• Subjects: Cell culture; Chromatography, Liquid - methods; Mass Spectrometry; Neurons - chemistry; Neuroscience; Phosphorylation; Protein Biochemistry; Proteins - analysis; Proteomics; Proteomics - methods; Protocol; Tandem Mass Spectrometry; Cell culture; Neuroscience; Protein Biochemistry; Mass Spectrometry; Proteomics
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.101063

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). [Display omitted] •Preparation and maintenance of rat primary-cultured neuronal cells•Detailed protocol on phosphoproteomics sample preparation from cortical neurons•Core steps of downstream (phospho-)proteomics data processing and statistical analyses 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.