Targeted Cross-Linking Mass Spectrometry on Single-Step Affinity Purified Molecular Complexes in the Yeast Saccharomyces cerevisiae.

Protein cross-linking mass spectrometry (XL-MS) has been developed into a powerful and robust tool that is now well implemented and routinely used by an increasing number of laboratories. While bulk cross-linking of complexes provides useful information on whole complexes, it is limiting for the probing of specific protein "neighbourhoods," or vicinity interactomes. For example, it is not unusual to find cross-linked peptide pairs that are disproportionately overrepresented compared to the surface areas of complexes, while very few or no cross-links are identified in other regions. When studying dynamic complexes along their pathways, some vicinity cross-links may be of too low abundance in the pool of heterogenous complexes of interest to be efficiently identified by standard XL-MS. In this chapter, we describe a targeted XL-MS approach from single-step affinity purified (ssAP) complexes that enables the investigation of specific protein "neighbourhoods" within molecular complexes in yeast, using a small cross-linker anchoring tag, the CH-tag. One advantage of this method over a general cross-linking strategy is the possibility to significantly enrich for localized anchored-cross-links within complexes, thus yielding a higher sensitivity to detect highly dynamic or low abundance protein interactions within a specific protein "neighbourhood" occurring along the pathway of a selected bait protein. Moreover, many variations of the method can be employed; the ssAP-tag and the CH-tag can either be fused to the same or different proteins in the complex, or the CH-tag can be fused to multiple protein components in the same cell line to explore dynamic vicinity interactions along a pathway.