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Click-linking: a cell-compatible protein crosslinking method based on click chemistry

ABSTRACT: We developed a novel approach to in situ crosslinking mass spectrometry (XL-MS) where the reaction is split into two sequential and orthogonal coupling events. The method involves pre-stabilization of the proteome using a fixation protocol, followed by a two-step process that begins with extensive labeling of surface-available lysines with click-chemistry compatible reagents modified with NHS Esters. The second step involves the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of the installed precursors, which generates crosslinks with high efficiency. We observed approximately a 20-fold increase in protein-protein interactions detected by this approach when compared to standard DSS crosslinkers, and our Click-linking strategy even outperformed enrichable crosslinkers such as PhoX.

INSTRUMENT(S): Orbitrap Astral, Orbitrap Ascend

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

TISSUE(S): Cell Suspension Culture, Epithelial Cell, Cell Culture

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Bruno Amaral

LAB HEAD: David C.

PROVIDER: PXD059118 | Pride | 2025-01-28

REPOSITORIES: pride

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			Action	DRS
	20230816_A549_1xDSS_LiveCells_HighpH_F01.raw	Raw
	20230816_A549_1xDSS_LiveCells_HighpH_F02.raw	Raw
	20230816_A549_1xDSS_LiveCells_HighpH_F03.raw	Raw
	20230816_A549_1xDSS_LiveCells_HighpH_F04.raw	Raw
	20230816_A549_1xDSS_LiveCells_HighpH_F05.raw	Raw

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Project description:The HOP2/MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes, and promotes the repair of DNA double strand breaks. The HOP2/MND1 dimer forms via the central, split coiled coils (CC1 and CC2) present in both proteins, and the resulting complex contains several flexible regions such as the hinge between the coils. To investigate the conformational flexibility of the heterodimer, important for understanding mechanistic details of HOP2/MND1 function, we studied the spatial relation of the complex partners and their domains in solution. We performed chemical cross-linking in combination with mass spectrometry (XL-MS) to generate distance restraints, which were subsequently used for molecular modeling. The final XL-MS workflow encompassed the use of cross-linkers with varying spacer arm lengths, quenching, digestion, size exclusion enrichment and HCD based LC-MS/MS detection prior to data evaluation. We applied and systematically tested two different homobifunctional amine-reactive crosslinkers (DSS-11.4 Å and BS2G 7.7 Å) and one zero-length heterobifunctional crosslinker (EDC). Crosslinked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein-protein docking for crosslink guided molecular modeling. Our miniaturized SEC approach reduced the required starting material and led to a high amount of crosslinked peptides allowing the analysis of replicates. The majority of the identified crosslinks was found in the coiled coil domains, indicating a parallel orientation of the interaction partners. Furthermore, flexibility of the C-terminal head domains of HOP2 and MND1 was observed. The experimentally derived distance constraints combined with an iterative comparative modeling approach not only confirm the elongated, open conformation predicted by the crystal structure of the Giardia lamblia Hop2-Mnd1 heterodimer, but further suggest the coexistence of a closed complex conformation in solution.

Project description:Crosslinking mass spectrometry (XL-MS) is emerging as a unique method at the crossroads of structural and cellular biology, uniquely capable of identifying protein-protein interactions with residue-level resolution and on the proteome-wide scale. With the development of crosslinkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable crosslinks), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-crosslinkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-crosslinkers have not enjoyed widespread use, and have yet to be employed for proteome-wide studies, because their products are challenging to identify, and an MS-cleavable photo-crosslinker has not yet been reported. Here, we demonstrate the synthesis and application of two heterobifunctional photo-crosslinkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil MS-cleavable linkage upon acyl transfer to protein targets. Moreover, these crosslinkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-crosslinking mass spectrometry (photo-XL-MS), both in extracts and in cellulo. These studies provide a partial interaction map of the E. coli cytosol with residue-level resolution. We find that photo-XL-MS has a propensity to capture protein-protein interactions, particularly involving low-abundance uncharacterized proteins, suggesting it could be a powerful tool to shed light on the “darker” corners of the proteome. Overall, we describe methods that enable the detection of protein quinary interaction networks in their native environment at residue-level resolution proteome-wide, and we expect they will prove useful toward the effort to explore the molecular sociology of the cell.

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Click-linking: a cell-compatible protein crosslinking method based on click chemistry

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