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Identifying direct interactome and substrates of human thioredoxins in specific cellular components with new cysteine-reactive enrichable chemical cross-linkers

ABSTRACT: Identifying direct substrates of enzymes in specific cell compartment is challenging in methodology, but crucial for revealing unique cellular processes in different subcellular locations. Here we developed a comprehensive live cell cross-linking coupled mass spectrometry strategy to identify direct interactome of redox enzymes in subcellular level. We chemically synthesized two new enrichable bis-vinyl sulfone chemical cross-linker ePDES1 and ePDES2 with alkyne group, and firstly established two-step IMAC enrichment method for high efficiency cross-linking peptide enrichment. Based on high quality ePDES1 and ePDES2 cross-linking spectra, our method achieved identifying hundreds of direct interacting proteins and substrates cross-linking to active cysteine sites of TXN1 both in cytoplasm and nuclei and TXN2 in mitochondria. It is interesting to simultaneously identify proteins cross-linked to TXN1 active Cys32 and Cys73 responsible for denitrosylation and transnitrosylation, respectively. Our method firstly identified direct interactome and substrates of oxidoreductases in subcellular level, providing resources for precisely studying redox homeostasis in specific cell compartment.

ORGANISM(S): Homo Sapiens

SUBMITTER: Long Zhang

PROVIDER: PXD055461 | iProX | Mon Sep 02 00:00:00 BST 2024

REPOSITORIES: iProX

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