With its unique capability for studying direct protein interactions in solution, cross-linking mass spectrometry (XL-MS) has become a powerful tool for elucidating structural topologies of large protein complexes. To establish this valuable technology for projects aiming to solve the architecture of protein complexes or validate protein interaction networks, we used disuccinimidyl sulfoxide (DSSO), a commercially available MS-cleavable cross-linker. DSSO was added at two steps of the affinity purification (AP) process: directly on-beads, while the protein complexes were bound, or in-solution on the eluted proteins. The success of DSSO crosslinking was assessed by running each fraction from the AP pipelines (wash, flow-through, eluate, leftover on beads) on SDS-PAGE. Cross-linked proteins migrate as a larger size band or a smear at the top of the gel. When to perform cross-linking appeared to depend on the type of AP performed. During the TAP-AP of Saccharomyces cerevisiae Rbp7, about a third of the cross-linked proteins came out in the flow-through after on-beads XL, another third in the SDS eluate, and the last third was left-over on the beads. On the other hand, during the HALO-AP of human SAP30L, there was no difference between in-solution and on beads XL. These different fractions were analyzed by XL-MS on an Orbitrap Fusion Lumos. The detected cross-linked peptides were visualized using xiView and cross-linked sites shown to be very similar. In summary, XL-MS is possible after one AP step, however, the presence of crosslinker might affect bait-antibody interactions as observed during TAP-AP, while AP processes based on ligand-enzyme interactions, such as Halo-AP, are impervious to it. To avoid loss of cross-linked proteins, all fractions of an AP process should be saved and assessed for high molecular weight cross-linked protein complexes. Several fractions may be combined to increase the amounts of XL protein for further XL-MS analysis.

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Journal Article

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Florens L, Hao Y, Zhang Y, Wen Z, Banks CAS, Washburn MP

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