Azide-A-DSBSO crosslinker

Azide-A-DSBSO crosslinker for Mass Spectrometry

This protocol only provides a guideline, and should be modified according to your specific need.

1. Prepare the Azide-A-DSBSO crosslinker solution

(1) Prepare stock solution: Prepare Azide-A-DSBSO crosslinker stock solution with a concentration of 40mM in anhydrous DMSO. Notice: Prepare immediately before use. Aliquot to avoid freeze-thaw cycles.

(2) Prepare working solution: Dilute the stock solution with PBS to prepare a working solution with a concentration of 1μM. Please adjust the concentration of the working fluid according to the actual situation, and prepare it now to avoid repeated freezing and thawing. Notice: The working solution must be prepared fresh on the day of the experiment.

Calculate the required volumes of reagents for the cross-linking reaction using the table below.

2. Prepare protein sample (purified or cell lysate)

(1) Prepare the protein sample in Cross-linking buffer buffer (50mM HEPES, pH 7.5). Ensure the sample does not contain amine-containing compounds (e.g., Tris, glycine, or ammonium salts) that would interfere with the NHS ester reaction.

(2) Adjust the protein concentration to 0.5-2mg/ml. For cell lysates, centrifuge at 17000×g for 15min at 4°C to remove debris and measure protein concentration using BCA or Bradford assay.

(3) Add DMSO to the mixture if necessary to ensure compatibility, keeping final DMSO concentration ≤5%.

3. Cross-linking reaction and analysis

(1) Add Azide-A-DSBSO crosslinker working solution and mix immediately by vortexing or pipetting.

(2) Incubate the reaction at room temperature (25°C) for 60 minutes with gentle agitation.

(3) Quench the cross-linking reaction by adding 1M Tris-HCl (pH 7.5) to a final concentration of 100mM. Mix thoroughly and incubate for 15 minutes at room temperature.

(4) Remove excess crosslinker using desalting columns (MWCO 6-8kDa) or dialysis against PBS.

(5) Reduce disulfide bonds by adding DTT to a final concentration of 10mM. Incubate at 37°C for 60 minutes (or 56°C for 30 minutes).

(6) Alkylate free thiols by adding iodoacetamide (IAA) to a final concentration of 20 mM. Incubate at room temperature in the dark for 30 minutes. Quench residual IAA by adding DTT to 5 mM final concentration and incubating for 15 minutes.

(7) Dilute the sample with 50mM HEPES, pH 7.5, to reduce sodium deoxycholate concentration to 1%. Add Lysyl Endopeptidase (LysC) at a 1:50 enzyme-to-protein ratio (w/w) and incubate at 37°C for 2 hours. Subsequently add trypsin at a 1:50 ratio (w/w) and incubate overnight (16 hours) at 37°C.

(8) For click chemistry enrichment using DBCO-Sepharose beads, Wash DBCO-beads (30-50μl slurry per sample) three times with PBS. Combine the digested peptide mixture with washed DBCO-beads. Incubate at room temperature for 2-4 hours with gentle rotation. Pellet beads by centrifugation (1000×g; 2min) and discard supernatant. Wash beads sequentially: 3×1ml PBS; 2×1ml 0.1% SDS in PBS; 2×1ml 6M urea in PBS; 3×1ml 100mM ammonium bicarbonate.

(9) Elute cross-linked peptides from beads using acid cleavage, incubate beads with 0.1% TFA (trifluoroacetic acid) or formic acid for 30 minutes at 37°C to cleave the acid-labile C-S bonds.

(10) Dry the eluted peptides in a concentrator. Reconstitute in 0.1% formic acid for LC-MS/MS analysis.

4. Precautions:

(1) For your safety and health, please wear a lab coat and disposable gloves when operating

(2) This protocol and all reagents are for scientific research use only.

References:

[1] Jiang P L, Zhu Y, Cai J, et al. Optimized In‐Solution and Gas‐Phase Chemistry Enables High‐Efficiency Interactome Mapping by DSBSO‐Based Cross‐Linking Mass Spectrometry[J]. Angewandte Chemie, 2025: e18355.

[2] Matzinger M, Kandioller W, Doppler P, et al. Fast and highly efficient affinity enrichment of Azide-A-DSBSO cross-linked peptides[J]. Journal of Proteome Research, 2020, 19(5): 2071-2079.