A data-independent acquisition-based global phosphoproteomics system enables deep profiling

Kitata, Reta Birhanu; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Pei-Yi; Chen, Bo-Shiun; Chang, Yun-Chien; Nesvizhskii, Alexey I.; Sung, Ting-Yi; Chen, Yu-Ju

doi:10.1038/s41467-021-22759-z

A data-independent acquisition-based global phosphoproteomics system enables deep profiling

Journal Article · Wed May 05 00:00:00 EDT 2021 · Nature Communications

DOI:https://doi.org/10.1038/s41467-021-22759-z· OSTI ID:1815690

^[1]; ^[2]; Tsai, Chia-Feng ^[3]; Lin, Pei-Yi ^[4]; Chen, Bo-Shiun ^[5]; Chang, Yun-Chien ^[5]; ^[6]; Sung, Ting-Yi ^[2]; ^[5]

Academia Sinica, Taipei (Taiwan). Inst. of Chemistry; OSTI
Academia Sinica, Taipei (Taiwan). Inst. of Information Science
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Biological Sciences Division
Academia Sinica, Taipei (Taiwan). Inst. of Chemistry
Academia Sinica, Taipei (Taiwan). Inst. of Chemistry; National Taiwan Univ., Taipei (Taiwan). Dept. of Chemistry
Univ. of Michigan, Ann Arbor, MI (United States). Dept. of Computational Medicine and Bioinformatics. Dept. of Pathology

Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve deep and robust quantitative phosphoproteomics profiling for minute amounts of sample, we here develop a global phosphoproteomics strategy based on data-independent acquisition (DIA) mass spectrometry and hybrid spectral libraries derived from data-dependent acquisition (DDA) and DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (<0.1 ng), accurate site localization and reproducible quantification (~5% median coefficient of variation). As a proof-of-concept, we use lung cancer cell lines and patient-derived tissue to construct a hybrid phosphoproteome spectral library covering 159,524 phosphopeptides (88,107 phosphosites). Based on this library, our single-shot streamlined DIA workflow quantifies 36,350 phosphosites (19,755 class 1) in cell line samples within two hours. Application to drug-resistant cells and patient-derived lung cancer tissues delineates site-specific phosphorylation events associated with resistance and tumor progression, showing that our workflow enables the characterization of phosphorylation signaling with deep coverage, high sensitivity and low between-run missing values.

View Accepted Manuscript (DOE)

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

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1815690

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 12; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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