Česká společnost pro hmotnostní spektrometrii

(CEEPC/IPM/CMSC 2022 - FrO-11)
Rapid ion mobility-resolved phosphoproteomics with dia-PASEF

Denys Oliinyk ¹, Florian Meier ^1 *

1. Functional Proteomics, Jena University Hospital, 07743 Jena, Germany

Abstrakt

Although protein phosphorylation is one of the best-studied post-translational modifications, cellular function and kinase-substrate relationships remain enigmatic for the vast majority of all identified modification sites. To decipher cellular signaling networks, functional mass spectrometry-based phosphoproteomics is an increasingly attractive strategy that benefits directly from further technological advances. Here, we explore the analytical merits of trapped ion mobility mass spectrometry and data-independent acquisition (dia-PASEF). Using an optimized data acquisition scheme, we quantified over 12,000 phosphopeptides in one hour from low sample amounts equivalent to ~20 ug protein extract per analysis without a spectral library. Strikingly, in 7 min gradients we still quantified about 80% of the class I sites with high accuracy and reproducibility. Our data shows that this is at least partly due to the ion mobility separation compensating for the increased spectral density at shorter gradients. We thus conclude that dia-PASEF offers great potential for scaling up phosphoproteomics both in terms of throughput and sensitivity.

* Korespondující autor: florian.meier-rosar@med.uni-jena.de