An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

Tsai, Chia-Feng; Zhao, Rui; Williams, Sarah M.; Moore, Ronald J.; Schultz, Kendall; Chrisler, William B.; Pasa-Tolic, Ljiljana; Rodland, Karin D.; Smith, Richard D.; Shi, Tujin; Zhu, Ying; Liu, Tao

doi:10.1074/mcp.ra119.001857

Title: An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

Journal Article · Sun May 03 00:00:00 EDT 2020 · Molecular and Cellular Proteomics

DOI:https://doi.org/10.1074/mcp.ra119.001857· OSTI ID:1638041

^[1]; Zhao, Rui ^[2]; Williams, Sarah M. ^[2]; Moore, Ronald J. ^[1]; Schultz, Kendall ^[1]; Chrisler, William B. ^[1]; Pasa-Tolic, Ljiljana ^[2];

^[1];

^[1]; Shi, Tujin ^[1]; Zhu, Ying ^[2];

^[1]

Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ~2500 proteins and precise quantification of ~1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.

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Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); NCI CPTAC; NCI EDRN; NCI IMAT; National Institute of General Medical Sciences

Grant/Contract Number:: AC05-76RL01830; U24CA210955; ACN15006-001; R21CA223715; P41 GM103493

OSTI ID:: 1638041

Report Number(s):: PNNL-SA-149183; 1535-9476; 1535-9484

Journal Information:: Molecular and Cellular Proteomics, Vol. 19, Issue 5

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 71 works

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59 BASIC BIOLOGICAL SCIENCES
Boosting ratio
automatic gain control (AGC)
iBASIL
ion injection time (IT)
single-cell proteomics
mass spectrometry
quantification
systems biology
HPLC
omics

Title: An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

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References (31)

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