Reverse phase liquid chromatography (RP-LC) is the mode of choice for crosslinking analysis because it offers the broadest peptide coverage range. RP separation using low flow systems such as the Thermo Scientific EASY-nLC 1200 System and UltiMate 3000 RSLCnano System offers ideal integration into the crosslinking workflow. The EASY-nLC 1200 features operational simplicity and high performance, while the UltiMate 3000 RSLCnano offers versatility and unsurpassed precision.
Crosslinking Mass Spectrometry
Understanding protein interactions
Crosslinking mass spectrometry analyzes protein-protein interactions that are “locked in place” to better understand how proteins affect biological processes such as signaling cascades, gene upregulation, and energy (ATP) production.
A majority of protein functions are determined by their interactions with other proteins and cellular components such as nucleic and fatty acids. It is via such interactions that biological processes commence, conclude, and change. Therefore, the study of protein interactions using crosslinking mass spectrometry is instrumental in understanding both healthy and disease states.
Studying protein function through interaction
Crosslinking mass spectrometry has emerged as a powerful technique for studying protein-protein interactions. During the process of crosslinking, chemical crosslinkers are used to chemically join components of interacting complexes. This is followed by liquid chromatography (LC) separation and identification by mass spectrometry (MS) analysis. This workflow enables the study of protein-protein interactions by maintaining the original interacting complexes.
The Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer is an ideal platform for crosslinking mass spectrometry analysis. The availability of multiple fragmentation techniques (CID, HCD, ETD, and EThcD) on the Orbitrap Fusion Lumos, and the ability to use these different fragmentations at different stages of MSn, together enable clear identification of crosslinked peptides and protein-protein interaction sites.
Thermo Scientific Proteome Discoverer Software offers a powerful means for analyzing crosslinking data: The new XlinkX node within the software features a fully integrated crosslink peptide search engine for crosslinking MS analysis. This node enables crosslinked peptide annotation with assignment of inter- and intra-crosslinked peptides and mono-adducts. It is also compatible with non-cleavable and MS-cleavable crosslinkers.