Figure 3. Endogenous THBS1 is C-mannosylated by hiPSCs.
A Chromatographic separation and MRM monitoring of proteolytic peptides derived from a THBS1 fragment containing TSR1 to 3 recombinantly expressed in HEK 293T cells. Peptides containing the consensus site for C-mannosylation (WxxW/C) with no, one or two mannoses, elute noticeably later than the two other TSR derived peptides and are detected at significantly lower signal intensities.
B EICs of characteristic fragment ions derived from the mono- (right panels) and di-C-mannosylated (left panels) TSR3 peptide from hiPS cell culture supernatants containing recombinant THBS1 (upper panels) as a positive control (spike-in) are compared with native samples of hiPS cell culture supernatants without addition of THBS1 (lower panels). EICs were transformed using the Savitzky-Golay smoothing in Skyline. In the native samples (no spike-in), the corresponding C-mannosylated TSR3 peptides were detected with low intensities at expected elution times, confirming the presence of endogenous C-mannosylated THBS1 in hiPSC cell culture supernatants.