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

ABSTRACT: A549 cells were incubated with N8-propargyl spermidine, N1-propargyl spermidine, or native spermidine. Protein conjugates formed in cellulo were analyzed as follows. After cell lysis, probe-protein adducts were subjected to bioorthogonal CuAAC ligation and affinity enrichment on NeutrAvidin beads. The enriched proteins were digested with trypsin, and the resulting peptides were labeled with TMT. Proteins that showed enrichment in samples treated with the propargyl analogs versus spermidine were designated as analog-binding proteins. This analysis revealed specific covalent binding of N8-propargyl spermidine to ALDH1A1. In a complementary peptide-level workflow, CuAAC ligation to azide-PEG3-biotin was followed by tryptic digestion, capture of biotinylated peptides bearing the probe or its metabolic products, and LC-MS/MS analysis. Dependent peptide analysis identified modification on the ALDH1A1 peptide SPCIVLADADLDNAVEFAHHGVFYHQGQCCIAASR (positions 274–308). Site-localization analysis placed the modification within the GQCC segment (positions 301–304), with nucleophilic Cys303 in the catalytic center being a highly probable site. Further experiments showed that the binding is ALDH1A1 activity dependent.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): A-549 Cell

SUBMITTER: Remigiusz Serwa

LAB HEAD: Remigiusz Serwa

PROVIDER: PXD070993 | Pride | 2026-05-28

REPOSITORIES: Pride

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			Action	DRS
	RS30_TMT12_2ul.raw	Raw
	RS30_TMT12_2ul_reinj1.raw	Raw
	RS33_TRP.raw	Raw
	checksum.txt	Txt
	txt_dependent_peptide_search.zip	Other

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