Fig. 1

Overview of sample preparation and instrument workflow for quantitative phosphoproteomics using single-shot LFQ and pHASED. Proteins were extracted from target cell lines and digested into peptides. Two hundred micrograms of digested peptides were prepared for enrichment. In pHASED, known concentrations of spike-in heavy labeled phosphopeptides were added to each sample prior to phosphopeptide enrichment. For both single-shot LFQ and pHASED enriched phosphopeptides were then injected into an Orbitrap Exploris 480. In pHASED, nLC-MS/MS was coupled with a FAIMS interface using four different compensation voltages (CVs; − 70 V, − 60 V, − 50 V and − 40 V). Figure created with BioRender.com