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

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.

Streamlined single-cell proteomics by an integrated microfluidic chip and data-independent acquisition mass spectrometry

“…Note that before each experiment, the cell chamber and reaction vessel were coated with bovine serum albumin (BSA) to minimize adsorptive losses of peptides. For subsequent liquid chromatography (LC)–MS/MS analysis, single-shot DIA-MS acquisition parameters, including isolation window, resolution, peptide amount, and LC–MS/MS gradient were optimized to enhance proteomic profiling coverage in low numbers of cells (“Methods”) 32 .…”

Meiotic DNA breaks activate a streamlined phospho-signaling response that largely avoids protein level changes

“…DIA overcomes this problem by co-fragmenting all of the peptides in pre-defined mass/charge windows. The resulting fragmentation spectra are highly complex and require more advanced algorithms and spectral libraries to resolve peptide sequences (Schubert et al, 2015), but DIA is able to achieve greater reproducibility and quantitative sensitivity than DDA (Bruderer et al, 2017; Selevsek et al, 2015), in particular for phosphoproteomics (Bekker-Jensen et al, 2020; Kitata et al, 2021). We designed a workflow that includes both methods to take advantage of their unique strengths.…”

Post-Translational Modifications of G Protein–Coupled Receptors Revealed by Proteomics and Structural Biology

“…Historically, these PTM types were discovered using metabolic labeling with radioactive probes or enzymatic methods, and PTM sites were deduced by site-directed mutagenesis ( Prihandoko et al, 2015 ; Lu and Fang, 2020 ). More recently, prominent advancements in mass spectrometry (Nguyen et al)-based proteomics allow for systematic analysis of protein PTM sites and abundances in cultured cells and tissues ( Olsen and Mann, 2013 ; Hansen et al, 2021 ; Kitata et al, 2021 ), which facilitates PTM profiling in various GPCR proteins. In this review, we provide an overview of PTM types, locations, crosstalk and dynamic regulation for different GPCR proteins that are characterized mainly with proteomic approaches.…”