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Abstract
Chemical cross-linking-mass spectrometry (CX-MS) combined with bioinformatics tools is increasingly being used to analyze large-scale protein–protein interactions. It has gained importance in studies in proteomics, lipidomics, in systems and structural biology. Recently it has gained importance in preparation of homogeneous antibody-drug conjugates, which has been described as “a pinnacle of such targeting efforts.” What makes these approaches exciting is that using the “Click” and Bertozzi protocols in vivo studies can be carried out successfully. Using CX-MS combined with cryo-EM, structures of protein complexes can now be probed at almost molecular resolution (upto 3 Å). Chemical crosslinking is useful in materials science, as well. Major advances in both mass spectrometric techniques and bioinformatics tools today allow one to identify cross-linked peptides with highconfidence and with more user-friendly approaches. Crucial to this is the ability to capture intermolecular crosslinking reliably. The use of a new small NHS-aryl azido heterobifunctional cross-linker based is described here, which picks intermolecular crosslinking better. Thus, Lysozyme has been crosslinked successfully as established by the ‘dimeric’ band observed in SDS-PAGE. its tryptic digestion, ‘zip tip’ enrichment, ESI-MS, MS/MS and the data generated analyzed using StavroX 3.6.0.1, a bioinformatics software, especially suited for determining intermolecular crosslinking.
Highlights
Chemical cross-linking-mass spectrometry (CX-MS) combined with bioinformatics tools is increasingly being used to analyze large-scale protein–protein interactions
It has been proposed that structures and functions of large protein complexes at the molecular or atomic level in dynamic situations can be studied by combining cryo-electron microscopy with crosslinking- mass spectrometry (CX-MS) [19]
It has been successfully used to crosslink Lysozyme as a ‘proof of concept’. This is done in two steps, i.e., via an initial incubation step, which is followed by the second step of photolysis (366 nm) using a 6 W TLC visualization UV lamp
Summary
Chemical cross-linking-mass spectrometry (CX-MS) combined with bioinformatics tools is increasingly being used to analyze large-scale protein–protein interactions. The ‘dimeric’ band is excised, trypsin digested and subjected to mass spectrometric analysis. We employed the above protocol with the twin aims of confirming whether our new small heterobifunctional crosslinker does bring about intermolecular crosslinking successfully and whether the same could be established using modern mass spectrometric methods (MALDI-MS, MS/ MS; ESI-MS) combined with StavroX 3.6.0.1.
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