Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition without the need for spectral libraries

Abstract

Quantitative phosphoproteomics has transformed investigations of cell signaling, but it remains challenging to scale the technology for high-throughput analyses. Here we report a rapid and reproducible approach to analyze hundreds of phosphoproteomes using data-independent acquisition (DIA) with an accurate site localization score incorporated into Spectronaut. DIA-based phosphoproteomics achieves an order of magnitude broader dynamic range, higher reproducibility of identification, and improved sensitivity and accuracy of quantification compared to state-of-the-art data-dependent acquisition (DDA)-based phosphoproteomics. Notably, direct DIA without the need of spectral libraries performs close to analyses using project-specific libraries, quantifying > 20,000 phosphopeptides in 15 min single-shot LC-MS analysis per condition. Adaptation of a 3D multiple regression model-based algorithm enables global determination of phosphorylation site stoichiometry in DIA. Scalability of the DIA approach is demonstrated by systematically analyzing the effects of thirty kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that specific protein kinases mediate EGF-dependent phospho-regulation.

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV

“…For DIA phosphoproteome data, to convert peptide-level output of Spectronaut into PTM site-level report, the Peptide Collapse Perseus plugin was used (https://github.com/AlexHgO/Perseus_Plugin_Peptide_Collapse) 35 . Phosphosites with less than 0.75 localization probability were excluded.…”

Global impact of phosphorylation on protein endurance

“…The "minor peptide group" was defined as Modified Sequences. In particular, the PTM localization option in Spectronaut v13 was enabled to locate phosphorylation sites 31,53 , with the probability score cutoff >0.75 31 , resulting Class-I peptides 4 to be identified and quantified. All the other settings in Spectronaut were kept as Default.…”

Quantitative Method for Assessing the Role of Lysine & Arginine Post-Translational Modifications in Nonalcoholic Steatohepatitis

“…We confirmed the presence of these succinylated and acetylated peptides by showing the highest abundant transitions found in DDA and DIA acquisitions of 'one-pot' acetyl-Lys and succinyl-Lys immunoaffinity enrichment from WT mouse liver were also found in complex Gnmt -/-liver lysate ( Figure S8, S9). Furthermore, for the tri-acetylated H4 K [8,12,16] peptide, the relative abundance of these transitions were constant between DIA acquisitions acquired on the Orbitrap Fusion Lumos and the Sciex TripleTOF 6600. These acquisitions were run on instruments located at two different research institutes, and yet yielded the same confident identifications of an acetylated and succinylated peptide respectively.…”

“…The added benefit of DIA based sampling to reduce missing data and thereby improve the consistency of peptide detection and quantitation promises the continued advancement for the MS-based study of low abundant PTMs. In addition, recently published algorithms PIQUed, 7 Peptide Collapse plugin for Perseus, 8 Inference of Peptidoforms (IPF), 9 and Thesaurus, 10 claim reduced false site localization and greater PTM recovery providing an alternative to the classical approach to elucidate PTM dynamics in complex biological systems and a more complete understanding of the complexity of cellular signaling, which we have harnessed. 4,5,9,[11][12][13] In proteins, methylation generally occurs on Arg and Lys residues with the ε-amino group of Lys being either mono, di, or trimethylated by protein lysine methyltransferases while Arg can be mono or dimethylated by protein arginine methyltransferases.…”