Phosphoproteome profiling using the isobaric carrier without the need for phosphoenrichment

Phosphorylation is a crucial component of signaling cascades in a cell. It controls various biological cellular functions such as cell growth and apoptosis. Due to the low stoichiometry of phosphorylaion, enrichment of phosphopeptide prior to LC-MS/MS is necessary for comprehensive phosphoproteome analysis and typical quantitative phosphoproteomic workflows are often limited by the amount of sample input. To address this issue, we developed an easy-to-establish, widely applicable and reproducible strategy to amplify phosphoproteome signals from a small amount of sample without phosphoenrichment step. Taking advantage of the multiplexing nature of isobaric labeling to achieve merged signal from multiple samples, and using a large amount of enriched phosphopeptides as a carrier, we were able to amplify a trace amount of phosphopeptide in the unpurified sample to an identifiable level and quantify it with the reporter ion intensity of the isobaric tag. Our result showed that >1400 phosphopeptide were quantified from 250 ng of tryptic peptides of cell extract. Through proof-of-concept experiments of our strategy, we distinguished 3 types of lung cancer cell line based on their quantitative phosphoproteome data and also identified phosphoproteome changes upon cellular perturbation achieved by drug treatment.