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

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

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1
Section of Islet Cell Biology and Regenerative Medicine , Joslin Diabetes Center and Harvard Medical School , Boston , Massachusetts 02215 , United States.
2
Department of Pathology, Immunology, and Laboratory Medicine , University of Florida , Gainesville , Florida 32611 , United States.

Abstract

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.

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