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.
[doi:10.25345/C54T6FF0H]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: TIMS ; PASEF ; data-independent acquisition ; scan mode ; TOF
Principal Investigators: (in alphabetical order) |
Matthias Mann, Proteomics and Signal Transduction Max Planck Institute of Biochemistry Am Klopferspitz 18 D-82152 Martinsried, N/A |
Submitting User: | patricias |
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Owner | Reanalyses | |
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