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U of Heidelberg Mass Spec Core - Cirksena et. al. Implications of ADAMTS16 C-Mannosylation on Optic Fissure Fusion

Targeted MS Experiment

 
Endogenous THBS1 is C-mannosylated by human induced pluripotent stem cells.
Permanent Link: https://panoramaweb.org/cirksena.url Share
Cirksena K, Hütte HJ, Shcherbakova A, Thumberger T, Sakson R, Weiss S, Jensen LR, Friedrich A, Todt D, Kuss AW, Ruppert T, Wittbrodt J, Bakker H, Buettner FFR. The C-mannosylome of human induced pluripotent stem cells implies a role for ADAMTS16 C-mannosylation in eye development. Mol Cell Proteomics. 2021 May 8:100092. doi: 10.1016/j.mcpro.2021.100092. Epub ahead of print. PMID: 33975020.
Data License: CC BY 4.0 | ProteomeXchange: PXD024117 | doi: https://doi.org/10.6069/52bt-0y70
  • Organism: Homo sapiens
  • Instrument: QTRAP 5500
  • SpikeIn: Yes
  • Keywords: hiPSC, TSR, thrombospondin, C-Mannosylation
  • Lab head: Falk F. R. Buettner Submitter: Roman Sakson
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).
Experiment Description
By spiking-in the digested peptides of the recombinant THBS1 fragment into the secretome sample, we accounted for possible retention time shifts or ion suppression effects caused by the specific matrix. After a blank run with 0.1% TFA, which ensured that no carryover signal was present, the secretome sample without recombinant spike-in was measured using scheduled MRM to detect endogenously C-mannosylated THBS1-peptides. Final scheduled MRM measurements were performed with 1.8 sec target scan time and 300 sec MRM detection window width.
Sample Description
A THBS1 fragment comprising TSR1 to 3 was cloned directly into the HindIII and XbaI sites of pSecTagB (Invitrogen), expressed in HEK 293T cells, purified, separated by SDS-PAGE and digested with AspN. Peptides were resolubilized in 20% ACN / 0.1% (v/v) TFA and incubated for 5 min at RT, then diluted 10-fold with 0.1% of TFA to gain a final concentration of 2% ACN. These peptides were used for MRM assay development.The secretome sample was obtained by precipitation of human induced pluripotent stem cell culture supernatants which were separated by SDS-PAGE and digested with AspN as described above. Only proteins migrating above the 70 kDa marker band were excised to remove low molecular weight proteins.
Created on 2/10/21, 8:44 AM

Targeted MS Runs

 
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Cirksena_Figure_3

 

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.