Fig. 4.

N-terminome analysis of CM of HEK293 cells overexpressing SPPL3. a Immunoblots of TCA-precipitated CM (left) and lysates of carbonate-washed membranes (right) of untransfected (ctrl) and SPPL3-V5 WT-transfected Flp-In™ T-REx™ HEK293 cells. Treatment with doxycycline (Dox) induces SPPL3 overexpression and leads to increased release of EXTL3 and POMK into the CM. b Volcano plots showing peptides detected following HYTANE enrichment of CM from doxycycline-treated and untreated Flp-In™ T-REx™ SPPL3-V5 HEK293 cells (three technical replicates). Samples were analysed in parallel on the indicated LC–MS systems. 2,817 and 1,608 unique N-terminal peptides are plotted, respectively, and labelling/filtering is performed as outlined in Fig. 2e with x = 30 aa. Dashed lines represent threshold values (± 2 SD for log2 difference and p = 0.05). c Schematic overview and mapping of neo-N peptides found to be changed in abundance in CM (see Fig. 2h). Arrow height indicates relative intensities (mean of doxycycline-treated samples) for membrane-proximal peptides changed upon SPPL3 overexpression