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

Anal Chem. 2019 May 7;91(9):5794-5801. doi: 10.1021/acs.analchem.9b00024. Epub 2019 Mar 15.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antiviral Agents / pharmacology
  • Humans
  • Interferon-gamma / pharmacology*
  • Interleukin-1beta / pharmacology*
  • Islets of Langerhans / cytology
  • Islets of Langerhans / drug effects
  • Islets of Langerhans / metabolism*
  • Phosphopeptides / metabolism*
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Staining and Labeling / methods*
  • Tandem Mass Spectrometry / methods*


  • Antiviral Agents
  • Interleukin-1beta
  • Phosphopeptides
  • Phosphoproteins
  • Interferon-gamma