Endogenous THBS1 is C-mannosylated by human induced pluripotent stem cells.

ABSTRACT: We used a human thrombospondin-1 (THBS1) fragment comprising thrombospondin type 1 repeats (TSRs) 1 to 3, which was recombinantly expressed in HEK 293T cells and digested with AspN to determine chromatographic retention times and to optimise MS parameters. We detected two peptides from TSR2 and TSR3 containing the consensus site for C-mannosylation in their non-, mono- and di-C-mannosylated state. These peptides – irrespective of C-mannosylation - were detected at less than 3 % of the intensity of two other THBS-derived peptides which do not contain a WxxW motif (Figure 3A). With the knowledge of the specific chromatographic retention times and the intensity patterns of fragment ions for the two peptides containing the WxxW sites for C-mannosylation from the recombinant THBS1, we now analysed a native sample of hiPSC-derived proteins from cell culture supernatants by MRM. Indeed we detected the most intense fragment ions for the peptide DACPINGGWGPWSPW with one or two C-mannoses at the expected elution times and with the expected intensity profiles (Figure 3B).

ORGANISM(S): Homo Sapiens

SUBMITTER: Roman Sakson  

PROVIDER: PXD024117 | panorama | Wed May 12 00:00:00 BST 2021

REPOSITORIES: PanoramaPublic