Combinatorial Electrostatic Collision-Induced Dissociative Chemical Cross-linking Reagents for Probing Protein Surface Topology

To ascertain more information on protein domain orientation and complex structure associations using chemical cross-linking, we have developed a combination of electrostatic collision-induced dissociative cross-linking reagents that differentially react with protein surfaces which are effectively analyzed by liquid chromatography-tandem mass spectrometry using ion trap multistage collision-induced dissociation. Implementing our original design and methodology based on disuccinimidyl-succinamyl-aspartyl-proline (SuDP) (Soderblom, E. J.; Goshe, M. B. Anal. Chem 2006, 78, 8059-8068. Soderblom, E. J.; Bobay, B. G.; Cavanagh, J.; Goshe, M. B. Rapid Commun Mass Specirom 2007, 21, 3395-3408.), disuccinimidyl-succinamyl-valyl-proline (SuVP) was synthesized. The SuDP and SuVP reagents are the same except for the valyl and aspartyl groups which provide a distinctive chemical feature to each reagent. When performing labeling reactions using various protein-to-cross-linker ratios at pH 7.5, the negatively charged SuDP and neutral SuVP were used to label bovine serum albumin and hemoglobin. After protein digestion, the resulting peptides were analyzed using four different ion trap LC/MS3 acquisition methods incorporating multistage CID. The more polar BSA surface resulted in a number of unique interpeptide and intrapeptide cross-links for each reagent whereas the less polarized surface of hemoglobin produced similar results for both reagents. Based on the identification of dead-end products (i.e., a crosslink modification containing a hydrolyzed end) for each protein, the aminolysis reactivity of each modified lysyl side chain revealed a preference for reacting with each reagent according to its local electrostatic surface environment. Overall, combinatorial application of SuDP and SuVP chemical labeling produces a set of unique interpeptide, intrapeptide, and dead-end crosslinked products that provides protein structural information according to its electrostatic surface topology which has the potential to be used to more comprehensively probe protein structure and dynamics.