Journal Article

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

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

2021

Overview

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. Phosphoproteomics can provide systematic insights into disease-associated cell signaling changes. Here, the authors present a sensitive workflow integrating library-based and direct data-independent acquisition approaches, and a hybrid spectral library resource for in-depth phosphoproteomic profiling.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/1647/296 / 631/45/475 / 631/67/1612/1350 / 631/80/458/1733 / 82/16 / 82/58 / 82/80 / 96/95 / Biotechnology / Cell Line, Tumor / Coefficient of variation / Depth profiling / Drug resistance / Humanities and Social Sciences / Humans / INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY / Libraries / Localization / Lung cancer / Lung Neoplasms - metabolism / Mass spectrometry / Mass spectroscopy / MATHEMATICS AND COMPUTING / multidisciplinary / Phosphopeptides - metabolism / Phosphorylation / Proteins - metabolism / Proteome - analysis / Proteomics / Science / Science (multidisciplinary) / Sensitivity / Signaling / Spectra / Tandem Mass Spectrometry - methods / Tumor cell lines / Workflow