In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker

Abstract

Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to obtain the dynamic conformations and interaction interfaces of protein complexes. Limited by the poor cell membrane permeability, chemical reactivity, and biocompatibility of crosslinkers, in vivo crosslinking to capture the dynamics of protein complexes with finer temporal resolution and higher coverage is attractive but challenging. In this work, a trifunctional crosslinker bis­(succinimidyl) with propargyl tag (BSP), involving compact size, proper amphipathy, and enrichment capacity, was developed to enable better cell membrane permeability and efficient crosslinking in 5 min without obvious cellular interference. Followed by a two-step enrichment method based on click chemistry at the peptide level, 13,098 crosslinked peptides (5068 inter-crosslinked peptides and 8030 intra-crosslinked peptides) were identified under the data threshold of peptide-spectrum matches (PSMs) ≥2 on the basic of the FDR control of 1%, which was the most comprehensive dataset for homo species cells by a non-cleavable crosslinker. Besides, the interactome network comprising 1519 proteins connected by 2913 interaction edges in various intracellular compartments, as well as 80S ribosome structural dynamics, were characterized, showing the great potential of our in vivo crosslinking approach in minutes. All these results demonstrated that our developed BSP could provide a valuable toolkit for the in-depth in vivo analysis of protein–protein interactions (PPIs) and protein architectures with finer temporal resolution.

Suborganelle-Specific Protein Complex Analysis Enabled by in Vivo Cross-Linking Coupled with Proximal Labeling

“…The long alkyl chain provides the lipophilicity and a wider reaction radius, while the o-hydroxy group was introduced to increase its water solubility. Previous work has shown that this trifunctional cross-linker BSP can achieve in-depth in vivo crosslinking in minutes; 18 therefore, we performed a head-to-head comparison with BSP to characterize the in vivo cross-linking S5). This indicates that DSDHD has suitable membrane permeability and crosslinking reactivity, while the longer spacer arm can enhance the possible benefits of systematic analysis for protein interactions (11.4 Å of BSP vs 16.0 Å of DSDHD).…”

Alkynyl-Enrichable Carboxyl-Selective Crosslinkers to Increase the Crosslinking Coverage for Deciphering Protein Structures

“…(A) Chemical structures of the trifunctional crosslinkers with two reactive groups marked in red and an enrichable handle shaded in orange. The maximal distance restraints between the Cα atoms of the two crosslinked acidic residues were calculated by setting all intervening dihedral angles to 180° using Chem3D 19.0 software . (B) MS/MS spectra of the crosslinked peptides by the three crosslinkers.…”

“…The peptide samples (8 mg for each) were dissolved in 4 mL of 0.1% sodium dodecyl sulfate (SDS)/PBS, and the click chemistry was carried out at 37 °C for 3 h by addition of 200 μL of 20 mM azo biotin-azide (ABA, in DMSO), 200 μL of 80 mM sodium l -ascorbate (Vc), 200 μL of 80 mM tris­(3-hydroxypropyltriazolyl-methyl) amine (THPTA), and 200 μL of 80 mM CuSO 4 in turn. After the reaction, the excess reagents Vc, THPTA, and CuSO 4 were removed using C18 tips and the excess ABA was removed using mix-mode cation exchange (MCX) columns (Waters Oasis, Ireland), as previously described . The resulting biotinylated peptides were subjected to enrichment using streptavidin agrose resin.…”

“…Therefore, it is still urgent to improve the crosslinking efficiency of crosslinkers targeting acidic residues. In addition, to achieve in-depth crosslinking analysis of complex samples, an enrichable tag is indispensable, − especially for the carboxyl-selective crosslinkers with limited reaction efficiency.…”

“…Three alkynylenrichable carboxyl-selective crosslinkers were synthesized through a two-step procedure starting from BSP, as depicted in Supporting Scheme S1. BSP is a kind of lysine-targeting crosslinker containing two NHS esters, 22 which makes it readily derivable into other functional molecules. The first step was the introduction of the t-butyloxycarboryl (Boc)-protected reactive groups (hydrazide, amino, or aminooxy) into BSP through the amidation reaction.…”