Optimized Protocol for Protein Macrocomplexes Stabilization Using the EDC, 1‑Ethyl-3-(3-(dimethylamino)propyl)carbodiimide, Zero- Length Cross-Linker Ele ́ onore Lepvrier, † Cyrielle Doigneaux, † Laura Moullintraffort, †,‡ Alexis Nazabal, § and Cyrille Garnier* ,† † Translation and Folding, UMR-CNRS 6290, Universite ́ de Rennes 1, 35042 Rennes Cedex, France § CovalX AG, 8952 Zü rich-Schlieren, Switzerland 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. A lmost all cellular functions require functional macro- complexes, resulting from noncovalent interactions of proteins. A comprehensive description of these interactions is essential to understand how these vital macrocomplexes control diverse cellular functions. 1 This way, several techniquesX-ray crystallography, nuclear magnetic resonance (NMR) spectros- copy, and cryo-electron microscopy (Cryo-EM)have been developed to investigate the structure of these complexes and to elucidate these protein-protein interactions. These techniques provide lots of information concerning proteins and the protein macrocomplexes structural organization. Nevertheless, each technique has its limits (i.e., protein quantities and concentrations, the ability to crystallize, the macrocomplex stability, the molecular weight, or the use of unphysiological conditions). To overcome these limits and to acquire complementary information, new techniques and methods are currently being developed. Over the last 20 years, because of its high sensitivity, mass spectrometry (MS) and high mass spectrometry (HMS) have been emerging and appear to be very promising for both the protein identification in macrocomplexes and the understanding of the macro- complexes organization. 2 Indeed, contrary to the other techniques, MS enables rapid analysis of minute amounts of purified protein, providing, in addition to the mass determi- nation, stoichiometric information and identification of the interacting domains inside macrocomplexes. Nevertheless, even if MS seems to be a powerful tool to study macrocomplexes, dissociation of protein complexes during purification and analysis is a major difficulty. 3 Since then, cross-linking these noncovalent complexes in order to stabilize them becomes a prerequisite before MS analysis. This way, the combination of chemical cross-linking and HMS is an attractive and reliable approach for mapping the topology of multiprotein complexes, to identify interacting domains and to determine stoichiometry of interaction. 4 Several laboratories and companies such as CovalX developed their own cross-linkers for rapid, specific, Received: July 11, 2014 Accepted: September 30, 2014 Published: September 30, 2014 Technical Note pubs.acs.org/ac © 2014 American Chemical Society 10524 dx.doi.org/10.1021/ac502561e | Anal. Chem. 2014, 86, 10524-10530