Boosting to Amplify Signal with Isobaric Labeling (BASIL) Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells

Comprehensive phosphoproteomic analysis of small populations of cells remains a daunting task due primarily to the insufficient MS signal intensity from low concentrations of enriched phosphopeptides. Isobaric labeling has a unique multiplexing feature where the total peptide signal from all channels (or samples) triggers MS/MS fragmentation for peptide identification, while the reporter ions provide quantitative information. In light of this feature, we tested the concept of using a boosting sample (e.g., a biological sample mimicking the study samples but available in a much larger quantity) in multiplexed analysis to enable sensitive and comprehensive quantitative phosphoproteomic measurements with <100 000 cells. This simple boosting to amplify signal with isobaric labeling (BASIL) strategy increased the overall number of quantifiable phosphorylation sites more than 4-fold. Good reproducibility in quantification was demonstrated with a median CV of 15.3% and Pearson correlation coefficient of 0.9S from biological replicates. A proof-of-concept experiment demonstrated the ability of BASIL to distinguish acute myeloid leukemia cells based on the phosphoproteome data. Moreover, in a pilot application, this strategy enabled quantitative analysis of over 20 000 phosphorylation sites from human pancreatic islets treated with interleukin-1 beta and interferon-gamma. Together, this signal boosting strategy provides an attractive solution for comprehensive and quantitative phosphoproteome profiling of relatively small populations of cells where traditional phosphoproteomic workflows lack sufficient sensitivity.