Q exactive plus hybrid quadrupol orbitrap mass spectrometer

Samples were measured on a Q Exactive Plus Hybrid Quadrupol-Orbitrap mass spectrometer (MS) coupled to an EASY-nLC 1000 UHPLC (Thermo Fisher Scientific) and analyzed with a 240 min gradient using Top 10 DDA method. Phospho-proteomic samples were analyzed with a 90 min gradient using Top 10 DDA method. Raw MS data files were analyzed using MaxQuant software (Max Planck Institute of Biochemistry, Martinsried, Germany) (Cox and Mann 2008 (link)). We used the Uniprot Mouse database (release November 2019) extended by the human β₁-AR (hADRB1) sequence for spectral matching. Default settings were used, and peptides and proteins were identified using a false discovery rate (FDR) of 1%. As variable modification, p(STY) was enabled. To appreciate the biological significance of the differentially phosphorylated proteins, the ingenuity pathway analysis (IPA, QIAGEN, Germany) was used to predict regulated ontology lists, “tox lists” and networks related to cardiovascular function and disease.

For the untargeted metabolite analysis, the collected phloem sap samples were diluted with 50% (v/v) methanol followed by ion chromatography separation using an ICS-5000+HPIC system (Thermo Scientific, Dreieich, Germany) coupled to a Q-Exactive Plus hybrid quadrupol-orbitrap mass spectrometer (Thermo Scientific, Dreieich, Germany). For details, see [19 (link)].