Dataset Information

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

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Publications

The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development.

Cirksena Karsten K Hütte Hermann J HJ Shcherbakova Aleksandra A Thumberger Thomas T Sakson Roman R Weiss Stefan S Jensen Lars Riff LR Friedrich Alina A Todt Daniel D Kuss Andreas W AW Ruppert Thomas T Wittbrodt Joachim J Bakker Hans H Buettner Falk F R FFR

Molecular & cellular proteomics : MCP 20210508

C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR-Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent st ...[more]

PMID: 33975020

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Project description:Two datasets: StREM1.3: methyl-beta cyclodextrin-dependent distribution of StREM1.3 to detergent resistant and detergent soluble membrane fractions. SLAH3: localization of SLAH3 to detergent resistant and detergent soluble fractions dependent on co-expression with CPK21 and ABI1. Data analysis: Protein identiﬁcation and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a ﬁxed modiﬁcation; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modiﬁcations. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identiﬁed by MaxQuant were excluded from further analys Protein identiﬁcation and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a ﬁxed modiﬁcation; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modiﬁcations. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identiﬁed by MaxQuant were excluded from further analysis. PXD000211 is also assoicated with the same study/paper.

Dataset Information

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

Publications

The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development.

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