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A New Heterobifunctional Cross-linker Based on an “Introverted” Acid: Mass Spectrometric and Bioinformatics Studies, Analysis of Intermolecular Crosslinking of Proteins

Sujeet Thakur K and Eswaran SV

A new NHS-aryl azido heterobifunctional cross-linker based on an “introverted” carboxylic acid has been used to bring about successful intermolecular cross-linking. As a ‘proof-of-concept’ Lysozyme was incubated with the crosslinker, then photolysed (366 nm, 6 W UV lamp), subjected to SDS-PAGE, excision of the ‘dimer’, trypsin digested and analyzed by ESI-MS and StavroX 3.6.0.1. Previous studies on crosslinking of Lysozyme (SI-I and SIII) using homobifunctional cross-linkers, either no cross-linking was observed or only two crosslinks were detected in the case of BS³, a smaller cross-linker. The heterobifunctional cross-linker described here leads to many more crosslinks, which have been identified by using mass spectrometry (ESI-MS) and StavroX 3.6.0.1, a bioinformatics software, especially suited for identifying intermolecular crosslinking.