Fast and Quantitative Recovery of Hydrophobic and Amphipathic Peptides after Incorporation into Phospholipid Membranes Lucie Khemte ´ mourian, Katell Bathany, Jean-Marie Schmitter, and Erick J. Dufourc* UMR 5144 MOBIOS, CNRS-Universite ´ Bordeaux 1, IECB, 33607 Pessac Cedex, France A new method that allows fast and quantitative recovery of hydrophobic or amphipathic peptides, or both, after their intimate incorporation into lipid membranes, is proposed. It relies on the use of small Sep-Pak cartridges and simple chromatographic handling. Peptides selected for this study are the 35 amino acid transmembrane domain of the Neu/erbB-2 protein and its point mutated (V664E) analogue expressed in some cancers, the 25 amino acid BH4 domain from the Bcl-2 antiapoptotic protein and the 15 amino acid Catestatin segment from chromogranin A found to have antimicrobial capabilities. Incorporation of peptides into membranes is accom- plished using organic solvent cosolubilization and several cycles of freeze-drying/hydration from aqueous solution. For the hydrophobic peptides, separation from the mem- brane is performed on Sep-Pak C 2 columns in two steps: (i) water/methanol elution of lipids and (ii) peptide elution using aprotic solvents (acetonitrile, 2-propanol). For amphipathic peptides, separation is performed on Sep- Pak C 18 columns using selective elution in one single step: water/methanol elution to recover first the peptide and then the lipids. Peptide and lipid recovery after all purification steps range from 60 to 80%, with peptide purity above 96%. This new method is simple, inexpen- sive, and very fast: a 10-mg membranous mixture con- taining 10% (w/w) peptide may be separated in 20-30 min. Syntheses of 15 N-labeled, 13 C-labeled peptides, or both are often difficult and very expensive. In particular, obtaining pure hydro- phobic peptides in good yields remains a challenging problem. 1-4 It is also known that studies of peptide-membrane interactions by biophysical techniques such as circular dichroism (CD), liquid- or solid-state NMR, or X-ray or neutron scattering require large amounts of material (20-40 mg). In such investigations, lipids may also be labeled and present in similar or greater quantities, henceforth increasing the experimental cost. It appears clearly that peptide and lipid recovery after their incorporation into model membranes such as bicelles membranes (bilayer micelles), small unilamellar vesicles, large unilamellar vesicles, or liposomes would be a significant benefit, owing to their value. To our knowledge, we found no published method to carry out the separation of peptides from lipidic membranes. Thus, we have designed a new method that makes it possible to separate and isolate both peptides and lipids quickly and in high yield. This method relies on the use of reversed-phase chromatographic supports packed in single- use cartridges (Sep-Pak, Waters); ensuring fast runs at low cost. Chromatographic conditions were selected in order to always adsorb peptides on the support, regardless of the lipid composi- tion, prior to their elution by means of a stepped-gradient elution. We chose a separation using single-use cartridges rather than HPLC equipment because it is simpler, faster, and less expensive. To work on a scale of ∼10 mg of lipids containing 10% (w/w) peptide, at least three injections would have been necessary for a separation by HPLC, with the use of a semipreparative column, and could take a day. With our new method, the same quantity of material may be purified at once in o.5 h and does not require specific equipment. To explore the versatility of our method, hydrophobic and amphipathic peptides were selected and their separation from different membrane model systems including bicelles and lipo- somes was studied. The first peptide chosen is derived from the single transmembrane part of the tyrosine kinase receptor neu/ erbB2. The sequence of the transmembrane peptide Neu TM35 is 650 EQRASPVTFIIATVVGVLLFLILVVVVGILIKRRR 684 . The sec- ond peptide corresponds to the naturally point-mutated analogue, Neu* TM35 , of sequence 650 EQRASPVTFIIATVEGVLLFLILVVVV- GILIKRRR 684 . The point of mutation occurring at position 664, a valine being replaced by a glutamic acid, promotes permanent triggering of cell growth, ultimately leading to death as in some cancers. 5-7 These peptides are largely hydrophobic with a small charged region and were incorporated into dimyristoyl- and dicaproylphosphatidylcholine bicelle membranes, which have attracted particular interest for biophysical studies (NMR, CD, * To whom correspondence should be addresed. E-mail: e.dufourc@ iecb.u-bordeaux.fr. Tel/fax/voice: +33 5 40 00 22 18. (1) Glover, K. J.; Martini, P. M.; Vold, R. R.; Komives, E. A. Anal. Biochem. 1999, 272, 270-274. (2) Goetz, M.; Rusconi, F.; Belghazi, M.; Schmitter, J. M.; Dufourc, E. J. J. Chromatogr.. B 2000, 737, 55-61. (3) Goetz, M.; Schmitter, J. M.; Geoffre, S.; Dufourc, E. J. J. Pept. Sci. 1999, 5, 245-250. (4) Khemte ´ mourian, L.; Lavielle, S.; Bathany, K.; Schmitter, J. M.; Dufourc, E. J. J. Pept. Sci. 2006, 12, 361-368. (5) Cao, H.; Bangalore, L.; Bormann, B. J.; Stern, D. F. EMBO J. 1992, 11, 923-932. (6) Chen, L. I.; Webster, M. K.; Meyer, A. N.; Donoghue, D. J. J. Cell. Biol. 1997, 137, 619-631. (7) Sanders, C. R.; Czerski, L.; Vinogradova, O.; Badola, P.; Song, D.; Smith, S. O. Biochemistry 1996, 35, 8610-8618. Anal. Chem. 2006, 78, 5348-5353 5348 Analytical Chemistry, Vol. 78, No. 15, August 1, 2006 10.1021/ac060207w CCC: $33.50 © 2006 American Chemical Society Published on Web 07/01/2006