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.
Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5800, USA.
Gwënaël
G
Pottiez
Pawel
P
Ciborowski
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D018260:Gelsolin / Q000097:blood / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D060066:Protein Interaction Maps; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D003432:Cross-Linking Reagents; D018260:Gelsolin