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 0.95 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-1β and interferon-γ. 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.