Enrichable cross-linkers for mapping direct protein interactions

Abstract

Background: It is crucial to investigate protein functions in specific subcellular environments. Cross-linking mass spectrometry is a powerful tool to map the direct interactome of proteins by identifying inter-protein cross-links. However, it is challenging to identify inter-protein cross-linked peptides due to their low abundance.

Results: We chemically synthesize the cross-linkers ePDES1 and ePDES2 with an alkyne group and a compound with azide linked to a phosphate group to enrich for cross-linked peptides.

Conclusion: Based on the high-quality cross-linking spectra of ePDES1 and ePDES2, our methods achieve the identification of hundreds of directly interacting proteins or substrates of thioredoxins in the nucleus and mitochondria.

Keywords: Cross-linking mass spectrometry; Direct protein interaction; IMAC; Subcellular organelles; Thioredoxin.

Fig. 1

Identifying direct interacting proteins and substrates of TXN1 with ePDES1 and ePDES2. a Chemical structures of ePDES1 and ePDES2. b Diagram representing processes that ePDES1 and ePDES2 trap substrates by cross-linking to active Cys32 and Cys73 residues of TXN1 in live 293 T cells. Western blot analysis showing ePDES1 (c) and ePDES2 (d) trapping multiple endogenous interacting proteins of TXN1 in live 293 T cell. Identifying ePDES1 (e) and ePDES2 (f) cross-linking peptides of purified TXN1 cross-linking complex before and after biotin enrichment. Number of ePDES1 (g) and ePDES2 (h) cross-linked peptides from TXN1 cross-linking complex of three replicates identified with XL-MS. i Types of cross-linking sites of ePSDES1 and ePDES2 cross-linking peptides. j Number of peptides and proteins cross-linked to active Cys73 and Cys32, respectively

Fig. 2

An IMAC enrichment method for enriching cross-linking peptides. a Chemical structure of phosphate-azide and diagram for phosphate labeling by click chemistry. Western blot analysis showing ePDES1 (b) and ePDES2 (c) trapped multiple endogenous interacting proteins of TPX in live E. coli cell. d Number of identified ePDES1 cross-linked peptides from TPX cross-linking complexes treated with or without lambda phosphatase. e Number of ePDES1 cross-linked peptides from TPX cross-linking complexes without lambda phosphatase treatment, common in three technical replicates. f Number of ePDES1 cross-linked peptides from TPX cross-linking complexes with lambda phosphatase treatment, common in three technical replicates

Fig. 3

IMAC enrichment of cross-linking peptides for identifying direct interactome and substrates of TXN1. a Diagram showing the workflow of XL-MS combined with IMAC enrichment and high pH fractionation. Number of ePDES1 (b) and ePDES2 (c) cross-linked peptides from TXN1 cross-linking complex of three replicates identified by XL-MS. Each replicate contained 4 fractions from high pH fractionation. d Types of cross-linking sites of ePDES1 and ePDES2 cross-linking peptides. e Number of TXN1 cross-linking peptides. f Number of peptides and proteins cross-linked to active Cys73 and Cys32, respectively. Overlap of identified proteins (g) and Cys sites (h) cross-linked to TXN1 Cys73 with BVSB or PDES, and ePDES1 or ePDES2 combined biotin and IMAC enrichment. i Comparison of identified TXN1 Cys73 cross-linking Cys sites by ePDES1 and ePDES2 with SNO database established by Cys-BOOST [35]. j ePDES1 and ePDES2 have advantages in identifying proteins cross-linked to TXN1 active Cys32, and 40 proteins were identified based biotin and IMAC enrichment

Fig. 4

Identify direct interactome of TXN1 with NLS sequence in live 293 T cells. a GO component analysis showing identified TXN1 interacting proteins by BVSB, PDES, ePDES1, and ePDES2 from cytosol and nucleus. b KEGG analysis of all identified interacting proteins of TXN1 with no NLS sequence. c Construct of TXN1 with nucleus location sequence NLS. Overlap of ePDES1 and ePDES2 cross-linked proteins (d) and Cys sites (e) of TXN1-NLS Cys73 with those identified using TXN1 Cys73 without NLS. f Comparison of identified TXN1-NLS Cys73 cross-linking Cys sites by ePDES1 and ePDES2 with SNO database established by Cys-BOOST. g GO component analysis of unique 101 TXN1-NLS interacting proteins. h Molecular functional analysis of unique 101 TXN1-NLS interacting proteins. i Tandem mass spectrum showing TXNIP Cys173 cross-linked to TXN1-NLS Cys73

Fig. 5

Identify direct interactome of mitochondrial TXN2 in live 293 T cell. Number of ePDES1 (a) and ePDES2 (b) cross-linked peptides from TXN2 cross-linking complex of three replicates identified by XL-MS. Each replicate contained 4 fractions from high pH fractionation. c Types of cross-linking sites of ePDES1 and ePDES2 cross-linking peptides. d Number of peptides and proteins cross-linked to active Cys90 and Cys37 residues of TXN2, respectively. e GO component analysis of unique 113 TXN2 active Cys90 cross-linked proteins. f KEGG analysis of all identified interacting proteins cross-linking to TXN2 Cys90. Tandem mass spectra showing known substrate PRDX5 active Cys100 cross-linked to TXN2 active Cys90 (g), and another important Cys204 residue of PRDX1 cross-linked to TXN2 Cys 90 (h)