2008 Proteomics Seminar Tour

Phosphoproteomics – technologies and application to the study of depolarization-dependent protein phosphorylation in nerve terminals

Martin R. Larsen¹, Nicolai Bache¹, George Craft², Mark Graham² and Phillip J. Robinson²

¹Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark.
²Cell Signalling Unit, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia.

Depolarization of neurons rapidly collapses the membrane potential leading to an influx of calcium into nerve terminals. This initiates the release of neurotransmitters from small synaptic vesicles in nerve terminals and also initiates the recycling of the empty synaptic vesicle (endocytosis) for reuse. Nerve terminals, synaptosomes, can be rapidly isolated from neurons, however due to contamination with e.g., mitochondria, further purificaiton using percoll gradients are necessary to achieve pure synaptosomes. Depolarization-dependent calcium influx activates a phosphatase calcineurin which triggers dephosphorylation of key endocytic proteins and activates endocytosis. It also stimulates calmodulin-dependent protein kinases to phosphorylate proteins involved in synaptic vesicle pools for exocytosis. Only a small number of phosphosites in these proteins have been identified and quantified after depolarization to date.

Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems using low amounts of starting material. We have previously developed highly selective and sensitive methods for enrichment of phosphorylated peptides from complex mixtures including TiO₂ chromatography (Larsen MR et al., MCP, 2006) and the SIMAC strategy (Thingholm TE et al., MCP 2007). In addition, we have shown that a significant amount of hydrophilic phosphopeptides are lost because they are not retained by normal reversed phase material. However we introduced graphite columns to capture these phosphopeptides for mass spectrometry. In this study we combined peptide quantification using iTRAQ, peptide pre-separation strategies, our selective phosphopeptide enrichment procedures and reversed phase and graphite chromatography’s to achieve comprehensive phosphoproteome analysis of living nerve terminals. In addition we have performed ETD on the synaptosomal preparation to further increase the phosphoproteome coverage.