OBJECTIVES: Chemical cross-linking coupled with mass spectrometry (CXMS) offers the distance constraints critical for building the structural model of protein and protein complexes and understanding dynamics of biological systems. Originally developed for protein structural models, CXMS has evolved into method for studying protein complex formation, ligand-induced conformational changes, and quantitative structural analysis using isotopically labeled cross-linkers. METHODS: In this study we tested the potential of isotopically labeled MS-cleavable cross-linker to track the stability of the therapeutic monoclonal antibodies. RESULTS: A novel isotopically labeled MS-cleavable cross-linker was synthesized, and its reactivity was successfully tested on peptide and protein standards. Further, the novel cross-linker was utilized to test the stability of selected therapeutical monoclonal antibodies, bevacizumab and trastuzumab, adopting the data-independent acquisition. CONCLUSION: This study reports the advantages of using combination of 13C isotopically labeled MS-cleavable cross-linkers and data-independent mass spectrometry analysis for the automated identification of cross-linked products and thus monitoring the structural rearrangement of protein structure.

CF Plus Chemicals Brno Czech Republic

Department of Biochemistry Faculty of Science Charles University Prague Czech Republic

Institute of Microbiology The Czech Academy of Sciences Vestec Czech Republic

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