Boosting to Amplify Signal with Isobaric Labeling (BASIL) Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells.
Anal Chem
; 91(9): 5794-5801, 2019 05 07.
Article
em En
| MEDLINE
| ID: mdl-30843680
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 <100â¯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 20â¯000 phosphorylation sites from human pancreatic islets treated with interleukin-1ß 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfopeptídeos
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Fosfoproteínas
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Coloração e Rotulagem
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Ilhotas Pancreáticas
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Interferon gama
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Interleucina-1beta
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Espectrometria de Massas em Tandem
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article