Understanding chemical reactivity for homo‐ and heterobifunctional protein cross‐linking agents

Chemical cross‐linking, combined with mass spectrometry, has been applied to map three‐dimensional protein structures and protein–protein interactions. Proper choice of the cross‐linking agent, including its reactive groups and spacer arm length, is of great importance. However, studies to understand the details of reactivity of the chemical cross‐linkers with proteins are quite sparse. In this study, we investigated chemical cross‐linking from the aspects of the protein structures and the cross‐linking reagents involved, by using two structurally well‐known proteins, glyceraldehyde 3‐phosohate dehydrogenase and ribonuclease S. Chemical cross‐linking reactivity was compared using a series of homo‐ and hetero‐bifunctional cross‐linkers, including bis(sulfosuccinimidyl) suberate, dissuccinimidyl suberate, bis(succinimidyl) penta (ethylene glycol), bis(succinimidyl) nona (ethylene glycol), m ‐maleimidobenzoyl‐ N ‐hydroxysulfosuccinimide ester, 2‐pyridyldithiol‐tetraoxaoctatriacontane‐ N ‐hydrosuccinimide and succinimidyl‐[( N ‐maleimidopropionamido)‐tetracosaethyleneglycol]ester. The protein structure itself, especially the distances between target amino acid residues, was found to be a determining factor for the cross‐linking efficiency. Moreover, the reactive groups of the chemical cross‐linker also play an important role; a higher cross‐linking reaction efficiency was found for maleimides compared to 2‐pyrimidyldithiols. The reaction between maleimides and sulfhydryl groups is more favorable than that between N ‐hydroxysuccinimide esters and amine groups, although cysteine residues are less abundant in proteins compared to lysine residues. Copyright © 2013 John Wiley & Sons, Ltd.