title: Optimized protocol for protein macrocomplexes stabilization using the EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, zero-length cross-linker.
abstract: Since noncovalent protein macrocomplexes are implicated in many cellular functions, their characterization is essential to understand how they drive several biological processes. Over the past 20 years, because of its high sensitivity, mass spectrometry has been described as a powerful tool for both the protein identification in macrocomplexes and the understanding of the macrocomplexes organization. Nonetheless, stabilizing these protein macrocomplexes, by introducing covalent bonds, is a prerequisite before their analysis by the denaturing mass spectrometry technique. In this study, using the Hsp90/Aha1 macrocomplex as a model (where Hsp denotes a heat shock protein), we optimized a double cross-linking protocol with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC). This protocol takes place in a two-step process: initially, a cross-linking is performed according to a previously optimized protocol, and then a second cross-linking is performed by increasing the EDC concentration, counterbalanced by a high dilution of sample and, thus, protein macrocomplexes. Using matrix-assisted laser desorption ionization (MALDI) mass spectrometry, we verified the efficiency of our optimized protocol by submitting (or not submitting) samples to the K200 MALDI MS analysis kit containing N-succinimidyl iodo-acetate, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester), suberic acid bis(N-hydroxysuccinimide ester), disuccinimidyl tartrate, and dithiobis(succinimidyl) propionate, developed by the CovalX Company. Results obtained show that our optimized cross-linking protocol allows a complete stabilization of protein macrocomplexes and appears to be very accurate. Indeed, contrary to other cross-linkers, the "zero-length" feature of the EDC reagent prevents overdetermination of the mass of complexes, because EDC does not remain as part of the linkage.
authors.affiliation: Translation and Folding, UMR-CNRS 6290, Université de Rennes 1 , 35042 Rennes Cedex, France.
authors.forename: Eléonore
authors.initials: E
authors.lastname: Lepvrier
authors.forename: Cyrielle
authors.initials: C
authors.lastname: Doigneaux
authors.forename: Laura
authors.initials: L
authors.lastname: Moullintraffort
authors.forename: Alexis
authors.initials: A
authors.lastname: Nazabal
authors.forename: Cyrille
authors.initials: C
authors.lastname: Garnier
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry*; D055550:Protein Stability; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: C515652:AHSA1 protein, human; D003432:Cross-Linking Reagents; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D005022:Ethyldimethylaminopropyl Carbodiimide