Crosslinking mass spectrometry (XL-MS) 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.
Crosslinking mass spectrometry has emerged as a powerful technique for examining protein-protein interaction. Cross linking for protein-protein interaction studies enable visualization of the interacting regions by letting researchers create distant maps within protein complexes or individual proteins. XL-MS can be used in parallel with high resolution techniques such as cryo-electron microscopy (cryo-EM) or X-ray crystallography to obtain structural information. Crosslinking experiments provide distance constraints between regions within the protein creating low resolution three-dimensional structure information or general topology of the protein structure. For protein complexes XL-MS can also be used to determine organization of protein complexes.
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.
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.
Today’s cutting-edge research pushes LC-MS to its limits. Obtaining high confidence insights to enable accurate resolution of subtle differences and avoid costly dead-ends is needed faster than ever before. The Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer surpasses these limits with new innovations that deliver the ultimate flexibility to expand experimental scope, and with built-in intelligence to ensure the highest data quality and confidence. One system provides maximum insights, so you productively go beyond today’s discovery.
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.
Fan Liu, PhD, Utrecht University, describes the use of crosslinking mass spectrometry and how it aids in the understanding of protein complexes. A recently developed novel search engine (XlinkX) for cross linking analysis, enabling analysts to search any database of unlimited database size, is also discussed, underscoring that crucial information of the structure and biology of many protein complexes is now within reach.