Quantitative Phosphoproteomics of Selective Autophagy Receptors
Selective autophagy enables degradation of specific cargo such as protein aggregates or organelles and thus plays an essential role in the regulation of cellular homeostasis. Cargo specificity is achieved on the level of autophagy receptors that concurrently bind the cargo and the autophagosomal membrane. Recent studies have demonstrated that selective autophagy is tightly regulated by posttranslational modifications of autophagy receptors, in particular protein phosphorylation. Phosphorylation of autophagy receptors by different kinases, including Tank-binding kinase (TBK1), can increase their affinity toward the cargo or autophagosomes and thereby regulate the specificity and activity of selective autophagy depending on the cellular condition.
Here, we report an approach for quantitative analysis of phosphorylation sites on autophagy receptors using mass spectrometry-based proteomics. In this protocol, GFP-tagged autophagy receptors are purified based on the high-affinity binding between GFP and GFP-Trap agarose. Interaction partners and background binders are subsequently removed by washes under denaturing conditions to obtain a pure fraction of the bait protein, thereby reducing the complexity of the analyzed sample. The bait protein is then digested on-bead, and peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The described approach permits systematic identification and quantification of phosphorylation sites on autophagy receptors and other autophagic components. In addition to phosphorylation, this protocol is suitable for investigating other posttranslational modifications, including protein ubiquitylation.
Key wordsAutophagy receptors Selective autophagy Phosphorylation Mass spectrometry-based proteomics SILAC
This work is supported by the German Research Foundation (Emmy Noether Program, BE 5342/1-1 and SFB 1177 on Selective Autophagy).
- 2.Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107–132. https://doi.org/10.1146/annurev-cellbio-092910-154005CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Cox J, Mann M (2011) Quantitative, high-resolution proteomics for data-driven systems biology. Annu Rev Biochem 80:273–299. https://doi.org/10.1146/annurev-biochem-061308-093216CrossRefPubMedGoogle Scholar