Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Base peak chromatograms are shown for select SAX fractions following high-resolution LC-MS/MS using an Orbitrap Fusion mass spectrometer: (D) fraction 3 (green); (E) fraction 6 (red); (F) fraction 10 (grey). Peptides were fragmented by HCD, with neutral loss of 98 amu from the precursor ions triggering EThCD (Ferries et al, 2017). Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot. The ptmRS node was used for phosphosite localisation. Analysis was performed on three independent biological replicates for each condition (NT or PHPT1 siRNA). 