Authors
Séverine Clavier1,2, Xiuxia Du, Sandrine Sagan3, Gérard Bolbach1,2, and Emmanuelle Sachon1,2
Organizations
- Laboratoire des Biomolécules, UMR7203 UPMC-ENS-CNRS-INSERM ERL1157, cc182, 4 place Jussieu,75252 Paris Cedex 05, France
- Plateforme de spectrométrie de masse et protéomique, IBPS-UPMC, cc41, 7-9 quai saint Bernard,75252 Paris Cedex 05, France
- Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, USA
Abstract
Cell penetrating peptides (CPPs) are attracting attention because of their ability to deliver biologically active molecules into cells. On their way they can interact with membrane and intracellular proteins. To fully understand and improve CPP efficiency as drug delivery tools, their partners need to be identified. To investigate CPP-protein complexes, chemical cross-linking coupled to mass spectrometry is a relevant method. With this aim, we developed an original approach based on two parallel strategies, an intact complex analysis and a bottom-up one, to have a global characterization of the cross-linked complexes composition as well as a detailed mapping of the interaction zones.
CovalX Technology Used (Click each option to learn more)
Outcomes
Monomeric actin (G-actin) lyophilized in Tris, ATP, and CaCl2 was dialyzed against a buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM DTT, 200 μM CaCl2, 200 μM ATP, 0.005% NaN3 (pH 8.0)). Stock solutions of CPP and actin were diluted in the same buffer to concentrations of 10 μM. A different buffer containing 20 mM HEPES and 150 mM NaCl was used for CPP and albumin. All samples were incubated for 15 minutes and then 50 mM of K100 (SBAT, SBBT, GBAT) was solubilized in DMF and added to create a 2 mM final concentration of cross-linkers. The cross linking reactions occurred over a period of 120 minutes at room temperature with general stirring. The reactions were quenched with Tris-base to reach a final concentration of 15 mM. To verify the specificity of the cross-linking reaction, (R/W)9 CPP (10 μM) was incubated with a commercial mixture of insulin (4 μM), cytochrome C (12 μM), and myoglobin (16 μM) with the K100 Stabilization Kit. The MALDI-TOF analysis was compared with and without the cross-linking kit and revealed that there were no discriminating peaks.
Source
https://doi.org/10.1016/j.euprot.2014.03.002