Reactivity and Applications of New Amine Reactive Cross-Linker for Mass Spectrometry Detection of Protein Complexes



Claudia Bich1, Stefanie Maedler1, Katja Chiesa1, Fabio DeGiacomo1, Nicolas Bogliotti1, and Renato Zenobi1


  1. Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland


Chemical cross-linking of proteins permits the stabilization of noncovalent complexes through introduction of covalent bonds. A crucial challenge is to find the fastest and most efficient cross-linkers in order to minimize reaction times and to handle delicate complexes. New cross-linkers were synthesized by introducing N-hydroxyphthalimide, hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole as leaving groups instead of the commonly used N-hydroxysuccimidyl moiety. With the use of matrix-assisted laser desorption ionization (MALDI) mass spectrometry, these new cross-linkers were then compared with the commercially available disuccinimidyl suberate (DSS) for covalent stabilization of the gluthatione-S-transferase (GST) dimer and of an antibody−antigen complex. They showed a better efficiency, generated about 30% more cross-linked complex, and reacted about 10 times faster than DSS. The reaction with the GST dimer was utilized to get information about their reaction efficiency and kinetics. Their ability to stabilize only specific protein complexes was verified by incubating them with a mixture of the proteins GST and ubiquitin. Finally, the cross-linkers were incubated with synthetic peptides to study the selectivity of the binding with various amino acid side chains. Not only lysine but also tyrosine was found to react with the newly synthesized cross-linker containing 1-hydroxy-7-azabenzotriazole as the reactive group.

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Complex Tracker


Protein complexes or peptides were reacted with an excess of each cross-linker (DSS, DPS, SBBT, and SBAT dissolved in dimethyl formamide (DMF) at 2mg/mL) to a total volume of 10 μL. The peptides were diluted using tetraethyl ammonium bicarbonate buffer (10 mM, pH 9) until 25 μM was reached. The proteins were diluted using sodium phosphate buffer (10 mM, pH 8) to a final concentration of 7 μM (GST) and 4μM/8μM (3E7/bPrP). The mixed samples and cross-linker were incubated at room temperature for 5 to 60 minutes. 1 μL of the cross-linked solution was mixed with 1 μL of matrix (sinapic acid (10 mg/mL) dissolved in acetonitrile/water (1:1, v/v) with 0.1% TFA. 1 μL of the final mixture was spotted on a MALDI sample plate. To test the specificity of the binding between the protein complex and the cross-linker, ubiquitin (5 μL, 30 μM) was added to the GST solution (5 μL, 15 μM) before being incubated with each synthetic cross-linker separately at room temperature for 120 minutes. To study the kinetics, the cross-linking progress at room temperature was analyzed at different incubation times by quenching the reaction, mixing it with the matrix and then spotting it on a MALDI plate. All samples were analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system. After analysis, the data was interpreted using the CovalX Complex Tracker software.

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