Eléonore Lepvrier1, Cyrielle Doigneaux1, Laura Moullintraffort1,3, Alexis Nazabal2, and Cyrille Garnier1
- Translation and Folding, UMR-CNRS 6290, Université de Rennes 1, 35042 Rennes Cedex, France
- CovalX AG, 8952 Zürich-Schlieren, Switzerland
- ZMBH Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany
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.
CovalX Technology Used (Click each option to learn more)
CovalX has designed a detection system specifically for higher mass proteins and protein complexes. The HM1 and HM2 use an ion conversion detector which greatly increases the sensitivity of the mass spectrometer. Higher mass ions: incident ions collide with a conversion dynode array which creates smaller secondary ions. Then, the secondary ions are reaccelerated into a SEM and detected with higher sensitivity because of their higher velocity. The use of these detector systems for high mass protein ions helps to prevent detector saturation and allow for better sensitivity and profiling.
Experiments were performed by obtaining 12 μM thick tissue samples from the brains of adult male Wistar rats. The tissues were mounted onto conductive glass slides, frozen for an hour and washed. Following washing, the tissue sections were placed in a bath of cold acetone for 30 seconds, removed and a bath of cold 95% ethanol for another 30 seconds and finally, placed in chloroform for 1 minute. Samples were compared using different procedures (standard, HFIP, Leinweber, tween and H2O2) before 0.5 μL of the specific matrix was added using a micropipette or an automated sprayer. Each sample preparation was analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.
Standard sample preparation: chloroform and ethanol washes, mixed with matrix of 20 mg/ml sinapic acid in acetonitrile:0.1% TFA (&;3, v/v)
HFIP sample preparation: addition of 20 mg/ml sinapic acid in pure HFIP onto washed tissue and then addition of recrystallization solution (20 mg/mL sinapic acid in acetonitrile:0.1% TFA (7:3, v/v))
Leinweber sample preparation: placement of tissue samples onto a droplet of 20 mg/ml sinapic acid in 90% ethanol that contained 0.5 % Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether and 0.1% TFA. The droplet was allowed to dry and a droplet of sonicated sinapic acid suspended in xylene was placed on top of the tissue sample before being dried in a vacuum desiccator. Finally, matrix droplets were applied again using 20 mg/mL sinapic acid solutions in 90% ethanol and 50% acetonitrile.
Tween sample preparation: the tissues were washed and a matrix solution (20 mg/ml sinapic acid in acetonitrile:0.1% TFA with a low concentration of polyethylene glycol sorbitan monolaurate) was added and allowed to dry.
H2O2 sample preparation: tissues were covered in a 3% solution of H2O2 before being incubated in a saturated vapor pressure chamber for 30 minutes and then dried in a vacuum desiccator. Finally, the samples were prepared using a matrix solution (20 mg/ml sinapic acid in acetonitrile:0.1% TFA (7:3, v/v)).