An Aptly Positioned Azido Group in the Spacer of a Protein Cross-Linker for Facile Mapping of Lysines in Close Proximity
✍ Scribed by Piotr T. Kasper; Jaap Willem Back; Maxime Vitale; Aloysius F. Hartog; Winfried Roseboom; Leo J. de Koning; Jan H. van Maarseveen; Anton O. Muijsers; Chris G. de Koster; Luitzen de Jong
- Publisher
- John Wiley and Sons
- Year
- 2007
- Tongue
- English
- Weight
- 363 KB
- Volume
- 8
- Category
- Article
- ISSN
- 1439-4227
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✦ Synopsis
Abstract
Cross‐links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross‐links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross‐linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine‐specific cross‐linker, bis(succinimidyl)‐3‐azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross‐linked peptides from BAMG‐treated cytochrome c. One pathway leads to cleavage of the cross‐linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross‐linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP‐induced reaction products. These can be isolated by reversed‐phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross‐link‐derived reaction products can be isolated and identified indicates that the mapping of cross‐links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.