PXD050922-1

PXD050922 is an original dataset announced via ProteomeXchange.

Title	An accessible workflow for high sensitivity proteomics using Parallel Accumulation - Serial Fragmentation (PASEF)
Description	Deep and accurate proteome analysis is crucial for understanding cellular processes and disease mechanisms, however, it is also challenging to implement in routine settings. In this protocol we combine a robust and accessible chromatographic platform with an equally robust and high-performance mass spectrometric set up, to enable routine yet in-depth proteome coverage for a broad community. This entails tip-based sample preparation and pre-formed gradients combined with trapped ion mobility - quadrupole - time-of-flight mass spectrometry. It enables parallel accumulation - serial fragmentation (PASEF), in which all ions are used but spectra are resolved by ion mobility. Combined with data-independent acquisition (dia-PASEF), it offers high peak sampling rates and near-complete ion coverage. Here we explain how to balance quantitative accuracy, specificity, proteome coverage, and sensitivity by choosing best PASEF and DIA method parameters. The protocol streamlines the liquid chromatography - mass spectrometry setup and enables PASEF method generation and evaluation for varied samples. Our py_diAID tool automatically generates acquisition schemes where isolation windows are optimally positioned on the mass-to-charge and ion mobility plane. Hands-on time is three hours, and simple biological projects can be performed in three days. The implementation shown here allows reproducible quantification of 7,300 proteins in a human cancer cell line in 21 min with quadruplicate single-shot injections and 27,000 phosphosites in 21 min from for phospho-enriched quadruplicates. Synchro-PASEF, a next-generation scan mode, currently offers proteomic depth comparable to that of dia-PASEF. When analyzed with AlphaDIA, it showed superior quantitative reproducibility owing to its drastically reduced cycle times and high sampling efficiency.
HostingRepository	MassIVE
AnnounceDate	2024-10-17
AnnouncementXML	Submission_2024-10-17_02:41:18.178.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Patricia Skowronek
SpeciesList	scientific name: Homo sapiens; common name: human; NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; acetylated residue; iodoacetamide derivatized residue; Phospho
Instrument	timsTOF Pro; timsTOF Ultra

Revision	Datetime	Status	ChangeLog Entry
0	2024-03-23 04:15:08	ID requested
⏵ 1	2024-10-17 02:41:18	announced

no publication

submitter keyword: TIMS, PASEF, data-independent acquisition , scan mode, TOF

Matthias Mann
contact affiliation	Proteomics and Signal Transduction Max Planck Institute of Biochemistry Am Klopferspitz 18 D-82152 Martinsried
contact email	mmann@biochem.mpg.de
lab head
Patricia Skowronek
contact affiliation	MPI
contact email	skowronek@biochem.mpg.de
dataset submitter

MassIVE dataset URI

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