Boosting to Amplify Signal with Isobaric Labeling (BASIL) Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells
- BATTELLE (PACIFIC NW LAB)
- Harvard Medical School
- University of Florida
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 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-1ß and interferon-gamma. Together, this signal boosting strategy provides an attractive solution to comprehensive and quantitative phosphoproteome profiling of relatively small populations of cells where traditional phosphoproteomic workflows provide insufficient sensitivity.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1530853
- Report Number(s):
- PNNL-SA-140381
- Journal Information:
- Analytical Chemistry, Journal Name: Analytical Chemistry Journal Issue: 9 Vol. 91
- Country of Publication:
- United States
- Language:
- English
Similar Records
Mass spectrometry-based proteomics for analysis of hydrophilic phosphopeptides
A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics