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

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

doi:10.1038/s41467-021-22759-z

Abstract

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.

Highlights

Phosphoproteomics can provide insights into cellular signaling dynamics
We report a global phosphoproteomics system (GPS) strategy based on data-independent acquisition (DIA)-MS with direct DIA and library-based computation mapping to high-quality hybrid spectral library derived from dependent acquisition (DDA) and DIA data
With fast data acquisition by the Orbitrap MS instrument, we show that a comprehensive hybrid phosphoproteomics spectral library resource can serve as a digital map to recover phosphopeptides in the m/z and retention time domains of DIA data

Summary

Introduction

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. 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. The GPS strategy using a single-shot DIA achieves deep quantification of 38,255 phosphosites (20,420 class 1 sites) with 95% unique phosphosites covered by the library-based approach. Application to cell lines and patient tissues further reveals advantages of significantly lower between-run missing values, especially for pTyr, and high sensitivity with deep coverage

Methods

Results

Conclusion