High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform

Mass spectrometry has transformed the field of cell signalling by enabling global studies of dynamic protein phosphorylation (‘phosphoproteomics’). Recent developments are enabling increasingly sophisticated phosphoproteomics studies, but practical challenges remain. The EasyPhos workflow addresses these, and is sufficiently streamlined to enable analysis of hundreds of phosphoproteomes at a depth of >10,000 quantified phosphorylation sites. Here we present a detailed and updated protocol that further ensures high performance in sample-limited conditions, while also reducing sample preparation time. By eliminating protein precipitation steps and performing the entire protocol including digestion in a single 96-well plate, we now greatly minimize opportunities for sample loss and variability. This results in very high reproducibility and low sample requirements of 200 μg of protein starting material or less. After cell culture or tissue collection, the protocol takes 1 d, whereas mass spectrometry measurements require 1 h per sample. Applied to glioblastoma cells acutely treated with EGF, EasyPhos quantified 20,132 distinct phosphopeptides from 200 μg protein in less than one day of measurement time, revealing thousands of EGF-regulated phosphorylation events.