Deep coverage single-shot phosphoproteomics and ion mobility-based elucidation of phosphosite isomers using PASEF-DDA and PEAKS Webinar

Webinar: Wednesday, June 17th, 2020

11:00 am Eastern Daylight Time

Presented by Prof. Dr. Stefan Tenzer, University Medical Center of the Johannes Gutenberg University Mainz, Germany

A detailed and highly resolved analysis of phosphorylation sites is crucial to elucidate cellular signalling mechanisms. While LC-MS has developed into the method of choice for phosphoproteomic analyses, the correct determination of phosphorylation site remains a challenge. Especially coeluting and isobaric phosphopeptide isomers, which are phosphorylated on different residues, are often impossible to resolve in classical LC-MS/MS analyses.  Using Parallel Accumulation Serial Fragmentation data dependent acquisition (PASEF-DDA) on a timsTOF Pro instrument, we show that ion mobility spectrometry (IMS) enables the separation of certain isobaric isomers based on their Cross Collision Section (CCS), as the position of the phosphogroup affects the ion geometry in the gas phase. To validate our findings, we applied our workflow towards the detailed phosphoproteomic characterisation of patient-derived osteosarcoma metastasis cell culture samples using a single-step TiO2 enrichment without further subfractionation. Data processing in PEAKS X+ enabled the identification of 27,768 phosphopeptides containing 4672 isobaric phosphopeptide pairs, of which 51.4% were coeluting. IMS enabled the separation of 19% of the coeluting isomer pairs, refining the view of the phosphoproteome.