CrosslinkingMassenspektrometrie

Crosslinking Mass Spectrometry (XL-MS) is an analytical technique used to study the three-dimensional structure and interactions of proteins. It involves chemically linking proteins that are in close proximity in a biological system. These crosslinked proteins are then digested into peptides, and these peptides, along with the crosslinker, are analyzed using mass spectrometry. The mass spectrometer can identify and quantify the mass of these modified peptides. By analyzing the mass-to-charge ratio of the crosslinked peptides, researchers can deduce which amino acid residues were in close proximity and thus infer the spatial arrangement of the protein or the interfaces of protein complexes.

The process typically begins with the addition of a crosslinking reagent to a biological sample. These reagents have reactive groups that can form covalent bonds with specific amino acid side chains, such as lysines or cysteines. The choice of crosslinker depends on the application, with options for cleavable or non-cleavable crosslinkers, and varying lengths and reactivities. After crosslinking, the proteins are usually denatured, digested by proteases, and then the resulting peptides are analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The characteristic mass shifts introduced by the crosslinker allow for the identification of crosslinked peptides.

XL-MS has become a powerful tool for mapping protein-protein interactions, determining the topology of protein complexes, and even providing insights into protein conformational changes. It complements other structural biology techniques like X-ray crystallography and NMR spectroscopy, particularly for large and dynamic protein assemblies that are difficult to study by these methods. The data generated by XL-MS can be used to build or refine structural models.