Dataset Information

An intelligent Boosting to Amplify Signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics

ABSTRACT: 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 (e.g., our recent boosting to amplify signal with isobaric labeling (BASIL) approach1) 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 systematically evaluated and optimized the BASIL approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of single cells. This improved, intelligent BASIL (iBASIL) approach enables comprehensive and quantitative single-cell proteomics analysis by carefully controlling the boosting-to-sample ratio and optimizing MS automatic gain control and ion injection time settings. Using standard LC-MS platforms, iBASIL enabled precise quantification of >1,000 proteins from 0.1 ng of peptide (equivalent to a single cell), or ~800 proteins from single FACS-isolated MCF10A cells. Moreover, coupling iBASIL to nanoscale fractionation enabled quantification of>1,438 proteins while readily separating 3 different cell lines using single-cell-equivalent peptides. We believe iBASIL has broad utility for comprehensive and precise quantitative single-cell proteomics for systems biology and biomedical research, as well as for comprehensive proteomic analysis of size-limited clinical specimens that cannot be readily accessed by regular proteomics platforms.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tao Liu

PROVIDER: PXD016057 | JPOST Repository | Thu Mar 05 00:00:00 GMT 2020

REPOSITORIES: jPOST

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			Action	DRS
	MCF7_126127_131boosting_01ng_5E5_IT1000_Exp1_4May19_Oak_Jup-19-02-04.raw	Raw
	MCF7_126127_131boosting_01ng_5E5_IT1000_Exp3_4May19_Oak_Jup-19-02-04.raw	Raw
	MCF7_126127_131boosting_01ng_5E5_IT1000_Exp4_4May19_Oak_Jup-19-02-04.raw	Raw
	MCF7_126127_131boosting_01ng_5E5_IT300_Exp3_4May19_Oak_Jup-19-02-04.raw	Raw
	MCF7_126127_131boosting_01ng_5E5_IT300_Exp4_4May19_Oak_Jup-19-02-04.raw	Raw

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

Tsai Chia-Feng CF Zhao Rui R Williams Sarah M SM Moore Ronald J RJ Schultz Kendall K Chrisler William B WB Pasa-Tolic Ljiljana L Rodland Karin D KD Smith Richard D RD Shi Tujin T Zhu Ying Y Liu Tao T

Molecular & cellular proteomics : MCP 20200303 5

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 p ...[more]

PMID: 32127492

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