Technical Note pubs.acs.org/ac
Optimized Protocol for Protein Macrocomplexes Stabilization Using the EDC, 1‑Ethyl-3-(3-(dimethylamino)propyl)carbodiimide, ZeroLength Cross-Linker Eléonore Lepvrier,† Cyrielle Doigneaux,† Laura Moullintraffort,†,‡ Alexis Nazabal,§ and Cyrille Garnier*,† †
Translation and Folding, UMR-CNRS 6290, Université de Rennes 1, 35042 Rennes Cedex, France CovalX AG, 8952 Zürich-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-Nhydroxysuccinimide 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.
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in macrocomplexes and the understanding of the macrocomplexes organization.2 Indeed, contrary to the other techniques, MS enables rapid analysis of minute amounts of purified protein, providing, in addition to the mass determination, 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,
lmost all cellular functions require functional macrocomplexes, 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 techniquesX-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, 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 © XXXX American Chemical Society
Received: July 11, 2014 Accepted: September 30, 2014
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dx.doi.org/10.1021/ac502561e | Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Technical Note
and sensitive MS analysis.5 Chemical cross-linking creates covalent bonds with variable length between lateral chains of protein amino acids in complexes. Among all the chemical cross-linkers available, carbodiimides are zero-length crosslinking reagents used to directly couple carboxyl groups to primary amines.4d,6 The N-(3-(dimethylamino)propyl)-N′ethylcarbodiimide hydrochloride (EDC) cross-linker is the most commonly used one. EDC mediates the formation of amide bonds between the negatively charged side chain carboxyl group (i.e., Glu or Asp) and the positively charged side chain amino group (Lys), when they are spatially close (200 client proteins and is known to selfassociate.8 Hsp90 interacts with a set of co-chaperones, among which Aha1 is the most potent activator of the low basal Hsp90s ATPase activity. Aha1, which is an elongated protein made of two domains, seems to favor the transition of Hsp90 from an open state to a closed state, thereby accelerating the chaperoning cycle and ATP hydrolysis.9 It has been widely demonstrated that Aha1 is able to bind Hsp90 dimer9d,10 but nothing is known about its interaction with the Hsp90 oligomeric species. In this study, we focused on complexes formed between Aha1 and the Hsp90 tetramer and demonstrated that our EDC cross-linking optimized protocol led to a complete macrocomplexes stabilization. Once stabilized, macrocomplexes became available for Cryo-EM, for MS (coupled or not) to trypsin digestion to identify interacting domains, and for HMS to determine the protein composition within macrocomplexes (i.e., molecular weight and stoichiometry). Efficiency of the EDC optimized cross-linking protocol was verified using HMS, after submitting (or not submitting) samples to the K200 cocktail of cross-linkers developed by the CovalX Company.
plasmid transformed in BL21 gold competent cells. Cultures were grown at 37 °C in Luria−Bertani medium containing 50 μg/mL kanamycin (Sigma−Aldrich) to reach an A600 nm of 0.6. Protein expression was induced with IPTG at a 0.5 mM final concentration for 3 h at 30 °C, cells were harvested and lysed by three passages at the French press in 100 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM imidazole and 5 mM DTT (lysis buffer). Lysates were clarified by ultracentrifugation at 25 000 rpm, for 30 min, at 4 °C (50.2 Ti, Beckmann) and loaded on a 10 mL HisTrapFF column conditioned in lysis buffer. Aha1 was eluted with 100 mM Tris-HCl pH 7.5, 100 mM NaCl, 250 mM imidazole and 5 mM DTT (elution buffer), by a linear gradient performed on an AKTAprime system (GE Healthcare). Fractions containing Aha1 were injected on a Superdex 200 26/40 (GE Healthcare) conditioned in 10 mM MES−NaOH (pH 6.5), the protein peak was pooled and concentrated. Purified samples were ultracentrifuged at 100 000g, for 30 min, at 4 °C, then flash-frozen in liquid nitrogen and stored at −80 °C. Aha1 concentration was determined by measuring UV absorption with molar absorption coefficient (280 nm) of 65 555 M−1 cm−1 in MES-NaOH buffer (pH 6.5). Cross-Linking Reactions. EDC Single and Double CrossLinking. Hsp90/Aha1 complexes (37.5 μM/37.5 μM or 37.5 μM/75 μM) were formed in 10 mM MES buffer, 13 mM MgCl2, 5 mM AMP-PNP (pH 6.5). The single cross-linking reaction was performed using N-(3-(dimethylamino)propyl)N′-ethylcarbodiimide hydrochloride (EDC) (Sigma−Aldrich) at a final concentration ranging from 2.3 mM to 4.6 mM for 30 min at 25 °C8a in 10 mM MES buffer (pH 6.5). The second cross-linking was performed by diluting from 5 to 100 times the prestabilized complex mixture in EDC at a final concentration ranging from 2.3 mM to 22.8 mM during 15 or 240 min at 25 °C or overnight at 4 °C. Cross-linking reaction was stopped by the addition of 2.2 M Tris-HCl, pH 7.5 quenching buffer (10% of the final volume). Samples were then concentrated on Amicon Ultra YM50 (Millipore) to reach a volume of 500 or 100 μL, ultracentrifuged at 110 000g, for 5 min, at 4 °C and analyzed using size exclusion chromatography (SEC). Efficiency of the EDC Double Cross-Linking Protocol. Samples prepared according the EDC cross-linking optimized protocol were submitted (or not submitted) to the K200 crosslinkers cocktail 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, (using matrix-assisted laser desorption ionization (MALDI), K200 MALDI MS analysis kit; CovalX, Zürich, Switzerland). Size Exclusion Chromatography. A volume of 100−500 μL of cross-linked sample (0.5−2.6 mg of cross-linked complex, depending on column size) was loaded on a Superdex 200 SEC column, 1 cm × 30 or 1 cm × 210 cm (90/60/30/30 cm Tricorn columns in series, GE Healthcare) conditioned in the 20 mM Tris-HCl, 8 mM MgCl2, 150 mM NaCl, pH 7.5 mobile phase, controlled by an HPLC pump system (625 LC System, Waters). Elution was conducted at a flow rate of 0.5 or 1 mL/ min at room temperature. The eluate was monitored at 280 nm (Model 2996 photodiode array detector, Waters). When samples were separated on the 210 cm configuration, fractions of 0.5 mL were collected between 50 mL and 95 mL. Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectroscopy (MALDI-TOF MS). Sample Preparation. Double cross-linked Hsp90/Aha1 complexes were separated by size exclusion chromatography−high-
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EXPERIMENTAL PROCEDURES Protein Sources. Hsp90 Purification. Hsp90 was purified from pig brains according to the method reported by Yonezawa et al.11 and modified by Garnier et al.12 Purified samples were ultracentrifuged at 100 000g, for 30 min, at 4 °C, then flashfrozen in liquid nitrogen and stored at −80 °C. Protein concentration was determined by measuring UV absorption with a molar absorption coefficient (280 nm) of 124 000 M−1 cm−1 in MES-NaOH buffer (where MES = 4-morpholineethanesulfonic acid), pH 6.5, considering that Hsp90 is a dimer. The absorption was corrected for light scattering using Beckman DU640B spectrophotometer software. Expression and Purification of the Aha1 Cochaperone. Human Aha1 was expressed from pET28a-Aha1 encoding B
dx.doi.org/10.1021/ac502561e | Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Technical Note
Figure 1. Optimization of a cross-linking (CL) protocol for the complete stabilization of the Hsp90 quaternary structures. (A) Elution profile of a 500-μL sample of Hsp90 single EDC (4.6 mM) (black) and double EDC cross-linked after sample dilution by a factor of 20 or 40 in EDC (11.4 mM) (gray and dashed gray, respectively). Arrow indicates the elution volume of blue Dextran 2000. (B) 7.5% SDS-PAGE analysis of Hsp90 noncross-linked (1), single cross-linked (2 and 3) and double cross-linked (4 and 5). The gel was stained using Coomassie Brilliant Blue.
3.25 kV. HM2 system using a secondary-ion acceleration region in order to improve the detection sensitivity of large macromolecules by accelerating secondary ions formed on a conversion dynode. Instrument was calibrated using the C450 MALDI MS Calibration Kit (CovalX). This calibration kit allows the calibration of the matrix-assisted laser desorption ionization−time of flight mass spectroscopy (MALDI-TOF MS) for HMS analysis up to 450 kDa. This calibration solution consists of a solution of monoclonal IgM COV1 (197 073.6; 246 342.0; 328 456.0, and 492 684.1 Da). For each sample, three spots were analyzed. Presented spectra correspond to the average of 200 laser shots. The MS data were analyzed using Complex Tracker analysis software.
performance liquid chromatography (SEC-HPLC). SEC eluted fractions from 72.0 to 74.5 mL were pooled and concentrated to reach 0.4−0.5 mg/mL, concentration was determined by the Bradford method,13 using an elaborated Hsp90 standard curve. Samples were diluted in the K200 cross-linking buffer (CovalX; phosphate buffer, pH 7 in distillated water (1:9, v/v) from 1/2 to 1/64 in a final volume of 10 μL (from 250 μg/mL to 6 μg/ mL). One microliter (1 μL) (250 ng to 6 ng) of each dilution was mixed to 1 μL of a sinapinic acid matrix (10 mg/mL) in acetonitrile/water (1:1, v/v), trifluoroacetic acid 0.1%. After mixing, 1 μL (125 to 3 ng) of each sample was spotted (in triplicate) on the MALDI plate (SCOUT 384; AnchorChip). After crystallization at room temperature, the plate was introduced in the MALDI mass spectrometer. The remaining 9 μL of the sample were subjected to cross-linking using the CovalX K200 MALDI MS analysis kit (CovalX AG, Zurich, Switzerland). Each sample was mixed with 1 μL of a dimethylformamide (DMF) solution of K200 stabilizer reagent (2 mg/mL). The K200 stabilizer reagent is a 1:1:1 mixture composed of 1,1′-(suberoyldioxy) bisazabenzotriazole (SBAT, di(3H-1,2,3]triazolo[4,5-b]pyridin-3-yl) octane-dioate), glutaroyldioxy bisazabenzotriazole (GBAT, di(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl) glutarate), and decanoyldioxy bisazabenzotriazole (DBAT, di(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl) decanedioate). After 1, 3, or 6 h of incubation at room temperature, samples were prepared for MALDI analysis, as were non-K200 overcross-linked samples. Data Acquisition. Samples were analyzed by high mass MALDI-TOF MS immediately after crystallization. The analysis was performed using the standard nitrogen laser and focusing on different mass ranges from 0 to 1 200 kDa. Laser power was adjusted for each mass spectrometric measurement in order to acquire data just above the threshold of detection. For the measurement of higher-molecular-weight protein complexes, a higher laser power was necessary for the data acquisition, mainly because of the lower concentration of macromolecules. High-mass MALDI mass spectra were obtained using a Model 4800 MALDI-TOF time-of-flight instrument (Applied Biosystems, Foster City, CA) equipped with a HM2 high-mass retrofit system (CovalX AG, Zürich, Switzerland). The instrument operated in linear and positive mode by applying an accelerating voltage of 20 kV and using a delay extraction of 150 ns. The HM2 system operated in the high mass mode with acceleration voltage set to 20 kV and gain voltage set to 2.85−
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RESULTS
Protocol Optimization To Stabilize Hsp90 and Hsp90/ Aha1 Macrocomplexes. In order to obtain a complete crosslinking reaction of high-mass complexes, our objective was to optimize the one-step cross-linking protocol that we used to characterize the Hsp90 oligomerization process.8 As we previously demonstrated, single EDC cross-linking protocol was soft, only inducing the stabilization of oligomeric states under native conditions, since monomeric species were observed by SDS-PAGE and MALDI-TOF MS.8b To prevent this reversibility, we overstabilized macrocomplexes with a second cross-linking step by increasing the EDC concentration coupled to a sample dilution to prevent from unspecific crosslinking. Optimal experimental conditions (incubation time, temperature, EDC concentration, sample dilution, etc.) were determined by comparison of SEC elution profiles, using the one step cross-linked Hsp90 oligomers SEC profile as reference.8a Our objective was to obtain similar areas under elution profiles and comparable species distribution, when compared to the one-step previously optimized cross-linking protocol.8b To illustrate this, an optimization of the sample dilution is presented in Figure 1A. All elution profiles displayed classical multimodal curves, showing that the Hsp90 complexes eluted between 8 mL and 13 mL. Moreover, areas under curves (AUCs) of profiles were equivalent, the number of species and their relative proportions remained almost identical, showing a difference of
