Elucidating protein inter- and intra-molecular interacting domains using chemical cross-linking and MALDI-TOF/TOF mass spectrometry

摘要

Among many methods used to investigate proteins/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 eukaryotic; 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 7.7Å 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 intra-molecular linking.