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

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.

Figure 1.

BASIL quantitative strategy. (a) The small amount of study samples and the much larger amount of boosting sample were individually labeled with different TMT tags. (b) The differentially labeled peptides appear as a single peak (identical m/z at MS1 level), which represents the sum of intensities from both the study and boosting samples. (c) Sequence information can be obtained after MS/MS fragmentation of the peptide backbone and quantification information on the study samples can be obtained from the intensities of the individual TMT reporter ions.

Figure 2.

Evaluation of the performance of the BASIL strategy. (a) TMT-10 channel assignment. (b) Signal distribution of all TMT-10 channels. (c) Summary of identified phosphopeptides and phosphorylation sites (class 1 phosphorylation sites indicate the site localization probability is higher than 0.75). (d) CV for the TMT channels 126, 127N, and 127C.

Figure 3.

Summary of quantitative phosphoproteome analysis of human islet samples. (a) The TMT-10 channel assignment for experimental sets A and B (Pn indicates the different patient; + and — signs indicate the human islets with and without cytokine treatment, respectively; Reference is the mix of all human islet samples; Boost is the EndoC-βH2 cells). (b) The overlap of identified phosphopeptides between TMT set A and B. (c) The summary of identified phosphopeptides and phosphorylation sites.

Figure 4.

Quantitative phosphoproteome analysis of human islet samples. (a) PCA readily separates human islet samples by cytokine treatment but not by the respective TMT set. (b) Unsupervised hierarchical cluster analysis of phosphoproteome of the human islet samples. (c) The enriched Reactome signaling pathways (top 15) for up-regulated phosphorylation sites after cytokine treatment. (d) Distribution of the abundance of phosphorylation sites of STAT1 (S727) in human islet samples before and after cytokine treatment.