Phosphorylation, a ubiquitous mechanism of cellular signaling, is one of the most common post-translational modifications involved in regulation of cellular processes. Dysregulation of kinase pathways is associated with various cancers including renal cell carcinoma (RCC). As number of mass spectrometry-based strategies to study phosphoproteome was developed, we aim to compare widely used strategies of phosphopeptide enrichment and data acquisition to gain as deep as possible insight in changes of phosphoproteome in two RCC cell lines, 786-0 and Caki-1, after treatment with sunitinib, a potent tyrosine kinase inhibitor that targets VEGF and PDGF receptors and is commonly used for RCC treatment. In our work we compare i) the use of two phosphopeptide enrichment methods based on Fe-NTA and TiO2 performance, (ii) data-dependent acquisition (DDA) and data-independent acquisition (DIA) methods for phosphoproteome analysis using timsTOF Pro 2 mass spectrometer (Bruker), iii) the outputs from the DIA data processing software Spectronaut 18.5 and DIA-NN 1.8.1, and iv) deregulation of kinase pathways in 786-0 and Caki-1 cell lines in response to the sunitinib treatment. In total, use of DIA strategy with data processing in Spectronaut 18.5 reached higher phosphoproteome coverage with 9,419 identified phosphopeptides compared to 6,007 with the data acquisition in DDA mode. Within the DIA dataset, 10.6% more phosphopeptides were identified in phosphopeptide samples enriched by Fe-NTA than in the samples enriched by TiO2. The number of phosphopeptides identified exclusively using Fe-NTA enrichment was five times higher than those identified exclusively after TiO2 enrichment. The number of identified tyrosine-phosphorylated peptides was comparable between enrichment methods, with only 2.5% more phosphopeptides identified with Fe-NTA than with TiO2 enrichment. Nevertheless, 86% of phosphopeptides was identified using both enrichment methods across both cell lines. Analysis of DIA data indicated decreased phosphorylations of key substrates of kinases involved in the VEGF signaling pathway, such as MAPK14, MAPK1, PTK2, and AKT1 after sunitinib treatment. Simultaneously, increased phosphorylation of PTK2 kinase and an increased abundance of AXL kinase were observed. In a summary, this work compared various methodological approaches for phosphoproteome analysis, highlighted the benefits of DIA acquisition for analysis of phosphopeptides and showed a deeper coverage of RCC phosphoproteome with Fe-NTA enrichment.

Supported by Ministry of Health of the Czech Republic (project NU22-08-00230), all rights reserved, by MEYS CR infrastructure project LM2023042 and European Regional Development Fund-Project "UP CIISB" (No. CZ.02.1.01/0.0/0.0/18_046/0015974), and by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102)—Funded by the European Union—Next Generation EU.