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Evaluation of N8-propargyl spermidine (Probe 1) as a substrate of deoxyhypusine synthase in cellulo

ABSTRACT: Two complementary experiments were performed to evaluate whether N8-propargyl spermidine is a substrate of deoxyhypusine synthase in cells. HEK293 and mouse embryonic fibroblast (MEF) cells were incubated with either N8-propargyl spermidine (Probe 1) or spermidine, lysed, and subjected to CuAAC ligation with azido-PEG3-biotin. In the MEF experiment, biotinylated proteins were enriched on NeutrAvidin agarose resin, digested on-bead with trypsin, and, following TMT labeling and LC-MS/MS analysis, the data were processed in MaxQuant and analyzed for intensity enrichment in samples derived from cells treated with Probe 1 versus spermidine. In the HEK293 experiment, proteins were first digested with trypsin, after which biotinylated peptides were enriched on NeutrAvidin agarose resin and analyzed by LC-MS/MS. During MaxQuant processing, the peptide-level data were searched for Probe 1-derived hypusine and deoxyhypusine modifications carrying the biotinylated triazole tag, as well as for Probe 1-derived thiotriazole adducts on cysteine residues.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Mef Cell, Hek-293 Cell

SUBMITTER: Remigiusz Serwa

LAB HEAD: Remigiusz Serwa

PROVIDER: PXD080059 | Pride | 2026-06-23

REPOSITORIES: Pride

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			Action	DRS
	RS-82-1_mix1-6.raw	Raw
	RS-82-1_mix1-6_2.raw	Raw
	RS_300825_IAA_treated_1.raw	Raw
	RS_300825_IAA_treated_2.raw	Raw
	RS_300825_no_IAA_1.raw	Raw

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Evaluation of N8-propargyl spermidine as a substrate of deoxyhypusine synthase in cellulo

Project description:Two complementary experiments were performed to evaluate whether N8-propargyl spermidine is a substrate of deoxyhypusine synthase in cells. HEK293 and mouse embryonic fibroblast (MEF) cells were incubated with either N8-propargyl spermidine or spermidine, lysed, and subjected to CuAAC ligation with azido-PEG3-biotin. In the MEF experiment, biotinylated proteins were enriched on NeutrAvidin agarose resin, digested on-bead with trypsin, and, following TMT labeling and LC-MS/MS analysis, the data were processed in MaxQuant and analyzed for intensity enrichment in samples derived from cells treated with N8-propargyl spermidine versus spermidine. In the HEK293 experiment, proteins were digested with trypsin, biotinylated peptides were enriched on NeutrAvidin agarose resin, and after LC-MS/MS analysis and MaxQuant processing, the peptides were examined for the presence of N8-propargyl spermidine thio-triazole adducts on cysteine residues.

2026-03-13 | PXD071477 | Pride

N8-propargyl spermidine as a chemical probe for sensing of ALDH1A1 activity in cells

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Evaluation of N8-propargyl spermidine and spermine as substrates of deoxyhypusine synthase in the in vitro hypusination of eIF5A and RIPK1

Project description:eIF5A is a well-established protein substrate of deoxyhypusine synthase (DHS), which also requires spermidine as a small-molecule substrate. At the end of 2024, a publication claimed that RIPK1 is an additional DHS substrate and that N8-propargyl spermidine can also serve as a DHS substrate. To evaluate these claims, eIF5A and RIPK1 were subjected to in vitro hypusination in the presence of DHS, deoxyhypusine hydroxylase (DOHH; the enzyme that hydroxylates the deoxyhypusine residue generated by DHS), and either N8-propargyl spermidine or spermidine. For reactions containing N8-propargyl spermidine, the samples were split: one portion was directly digested with trypsin, whereas the other was subjected to CuAAC ligation with azido-PEG3-biotin prior to tryptic digestion. Following LC-MS/MS analysis and MaxQuant processing, the data were searched for peptides bearing hypusine, deoxyhypusine, and their N-propargylated analogs. In samples that underwent CuAAC ligation, additional searches were performed for PEG3-biotin-triazole-modified N8-propargyl-spermidine-derived hypusine and deoxyhypusine, as well as for cysteine-N8-propargyl spermidine thio-triazole adducts.

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2025-09-22 | PXD066655 | Pride

HeLa cell protein interactome of polyamine affinity-based probes.

Project description:HeLa cells were incubated with affinity-based probes designed as synthetic analogs of endogenous metabolites: putrescine, spermidine, and spermine, while a monoamine compound served as a control. Probe-protein interactions were captured in situ using UV-induced crosslinking. After cell lysis, biorthogonal ligation and affinity purification of the probe-conjugated proteins were carried out. These proteins were digested with trypsin, and the resulting peptides were labeled with TMT. Proteins that showed enrichment in samples treated with polyamine probes, compared to those treated with the monoamine control, were identified as polyamine-binding proteins.

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