title: Understanding chemical reactivity for homo- and heterobifunctional protein cross-linking agents.
abstract: 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.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093, Zürich, Switzerland.
authors.forename: Fan
authors.initials: F
authors.lastname: Chen
authors.forename: Simone
authors.initials: S
authors.lastname: Nielsen
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D021122:Protein Subunits; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D008301:Maleimides; D021122:Protein Subunits; D011506:Proteins; D013388:Succinimides; D013438:Sulfhydryl Compounds; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012260:Ribonucleases; C022542:ribonuclease S; C001426:N-hydroxysuccinimide
keywords: 2-pyridyldithiol; MALDI mass spectrometry; N-hydroxysuccinimide (NHS) esters; amine-to-sulfhydryl cross-links; chemical cross-linking; maleimides