Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward 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 an AssayMAP Bravo platform to meet these demands.