Robust, sensitive and automated phosphopeptide enrichment optimized for low sample amounts applied to primary hippocampal neurons

Due to the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting towards an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive and less prone to variability. Here, we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on a AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 µg of peptide starting material. The optimized workflow proved to be efficient, sensitive and reproducible, identifying, localizing and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications we monitored EGF-induced signaling in hippocampal neurons, starting with only 200,000 primary cells resulting in approximately 50 µg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.