Dataset Information

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

ABSTRACT: Quantitative phosphoproteomics has in recent years revolutionized understanding of cell signaling, but it remains a challenge to scale the technology for high-throughput analyses. Here we present a rapid and reproducible phosphoproteomics approach to systematically analyze hundreds of samples by fast liquid chromatography tandem mass spectrometry using data independent acquisition (DIA). To overcome the inherent issue of positional phosphopeptide isomers in DIA-based phosphoproteomics, we developed and employed an accurate site localization scoring algorithm, which is incorporated into the Spectronaut software tool. Using a library of synthetic phosphopeptides spiked-in to a yeast phosphoproteome in different ratios we show that it is on par with the top site localization score for data-dependent acquisition (DDA) based phosphoproteomics. Single-shot DIA-based phosphoproteomics achieved an order of magnitude broader dynamic range, higher reproducibility of identification and improved sensitivity and accuracy of quantification compared to state-of-the-art DDA-based phosphoproteomics. Importantly, direct DIA without the need of spectral libraries performed almost on par with analyses using specific project-specific libraries. Moreover, we implemented and benchmarked an algorithm for globally determining phosphorylation site stoichiometry in DIA. Finally, we demonstrate the scalability of the DIA approach by systematically analyzing the effects of thirty different kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that a large proportion of EGF-dependent phospho-regulation is mediated by a specific set of protein kinases.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Dorte Bekker-Jensen

LAB HEAD: Jesper V. Olsen

PROVIDER: PXD014525 | Pride | 2020-05-19

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20100113_092014_pilotExp-directDIA-LocProb-0-75_Report.txt	Txt
	20100113_125451_pilotExp-directDIA-LocProb-0-75.sne	Other
	20171001_QE3_nLC7_DBJ_SA_DIAphos_2s_15k_15min_01.raw	Raw
	20171001_QE3_nLC7_DBJ_SA_DIAphos_2s_15k_15min_02.raw	Raw
	20171001_QE3_nLC7_DBJ_SA_DIAphos_2s_30k_15min_01.raw	Raw

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

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