Elucidating protein inter- and intramolecular interacting domains using chemical cross-linking and matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry.

Among many methods used to investigate protein/protein interactions, chemical cross-linking combined with mass spectrometry remains a vital experimental approach. Mapping peptides modified by cross-linker provides clues about proteins' interacting domains. One complication is that such modification may result from intra- but not intermolecular interactions. Therefore, for overall data interpretation, a combination of results from various platforms is necessary. It is postulated that the secretory isoform of gelsolin regulates several biological processes through interactions with proteins such as actin, fibronectin, vitamin D-binding protein, and unidentified receptors on the surface of eukaryotes; it also has been shown to self-assemble eventually leading to the formation of homo-multimers. As such, it is an excellent model for this study. We used four cross-linkers with arm length ranging from 7.7 to 21.7Å and MALDI-TOF/TOF mass spectrometry as the analytical platform. Results of this study show that MALDI-based mass spectrometry generates high quality data to show lysine residues modified by cross-linkers and combined with existing data based on crystallography (Protein Data Bank, PDB) can be used to discriminate between inter- and intramolecular linking.