Universität zu Köln

Hünemeyer, Leon (2026). Development of new CID cleavable cross-linking reagents suitable for structure elucidation of proteins via MS. PhD thesis, Universität zu Köln.

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Abstract

This work deals with the synthesis and characterization of a series of cleavable cross-linking reagents, which are designed to react with protein moieties aiming for 3-dimensional structure elucidation of large biomolecules by MS. One group of cross-linking reagents are amine reactive cleavable cross-linkers designed for the modification protein lysine residues. Furthermore, they are designed for enrichment via immobilized metal affinity chromatography aiming to selectively enrich low abundant cross-links prior to investigation by tandem MS. In addition to the synthesis the cross-linking efficiency of all reagents is demonstrated using test systems on peptide and protein level. The reagents are compared to established reagents which are tested under the same conditions. Finally, the enrichment step is tested using the most promising cross-linkers and compared to cross-linking samples from the same reagent which were not enriched. In addition, this work contains a series of studies aiming for tryptophan side chain modification in proteins. It represents a pioneering study aiming to selectively modify the indole function of tryptophane side chains using cross-linking reagents. These reactions are based on CH activation of the tryptophane indole function using transition metal catalysts. The reactions are performed on tryptophane test systems. Finally, the work contains a study which is aligned to an established publication which used the cross-linker 1,1-carbonyl diimidazole CDI as a potent zero length cross-linking reagent. This study aimed to surpass the cross-linking efficiency of the published reagent with the ones that were investigated in this work. This was achieved with the help of the cross-linking reagents S,S-di(pyridin-2-yl) carbonodithioate and di(pyridin-2-yl) carbonate with great success on the test protein Bovine Serum Albumin (BSA).