期刊
ANALYTICAL CHEMISTRY
卷 91, 期 9, 页码 5794-5801出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.9b00024
关键词
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资金
- NIH from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [UC4 DK104167, DP3 DK110844, UC4 DK104197]
- National Institute of Allergy and Infectious Diseases (NIAID) [P01 AI42288]
- National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) [U24CA210955, U01 CA214116]
- National Institutes of General Medical Sciences (NIGMS) [P41GM103493]
- DOE [DE-AC05-76RL0 1830]
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.
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