A Signal Amplification via Boosting with Isobaric Labeling Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells

ABSTRACT: Despite recent advances in MS-based proteomics, comprehensive phosphoproteomics analysis of small populations of cells remains a daunting task, due primarily to the general lack of sufficient MS signals from such limited amounts of samples. In light of the unique multiplexing feature of isobaric labeling where the “total” peptide signal from all channels (or samples) triggers MS/MS for peptide identification while the reporter ions are providing quantitative information, we tested the concept of using a “boosting” sample (e.g., a biological sample mimicking the study samples) in such multiplexed analysis to enable sensitive and comprehensive quantitative phosphoproteomics measurements in <100,000 of cells. Indeed, this simple yet effective signal amplification via boosting in isobaric labeling (SABIL) strategy increased the number of quantifiable phosphorylation sites by more than 4-fold and demonstrated good reproducibility in quantification with a median CV of 15.3% and Pearson correlation coefficient of 0.95 from biological replicates. A proof-of-concept experiment also demonstrated the ability of SABIL to differentiate different acute myeloid leukemia cells based on the phosphoproteome data acquired therein. Furthermore, in a pilot application this strategy enabled quantitative analysis of over 20,000 phosphorylation sites of human islet samples from individual donors following cytokine treatment. Together, this signal boosting strategy provides an attractive solution to comprehensive and quantitative phosphoproteomic characterization of relatively small populations of cells where traditional phosphoproteomics workflow falls short of sensitivity.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tao Liu and Wei-Jun Qian 

PROVIDER: PXD011857 | JPOST Repository | Fri Mar 15 00:00:00 GMT 2019

REPOSITORIES: jPOST