title: Reaction of naphthalene-2,3-dicarboxaldehyde with enkephalins for LC-fluorescence and LC-MS analysis: conformational studies by molecular modeling and H/D exchange mass spectrometry.
abstract: A new labeling method compatible with both laser-induced fluorescence (LIF) and MS detection for enkephalins, which uses naphthalene-2,3-dicarboxaldehyde (NDA) and a new nucleophilic agent (N,N-dimethylaminoethanethiol) is described. When the derivative is separated via reverse phase HPLC and detected via MS, two different peaks with similar exact mass but different fluorescence and fragmentation properties are obtained. To interpret these results, molecular modeling and H/D exchange mass spectrometry studies were investigated to test the hypothesis that the peak obtained by LC/LIF/MS analysis depends on the site of protonation of the labeled enkephalins. The peptides labeled with NDA and N,N-dimethylaminoethanethiol were separated on a reverse phase C18 column with a gradient of aqueous 0.1% formic acid and acetonitrile. In mass spectrometry, two peaks are observed with the same exact mass for each molecule while only one peak is detected using fluorescence. Tandem mass spectrometry experiments of ion m/z 809.5 were performed on each chromatographic peak; the first peak (which is not observed by LIF detection) gives a fragment corresponding to the loss of the aminothiol side chain while no fragmentation is observed on the second peak, which was detected by fluorescence. The hypothesis is that each peak represents the labeled enkephalin with different sites of protonation. According to this hypothesis, three fundamental conformations that were closed to the unlabeled leucine-enkephalin were obtained by molecular modeling: a beta-turn like conformation with two hydrogen bonds, a 3(10)-helix with an H bond, and finally, the extended form without any intramolecular interactions. H/D exchange mass spectrometry experiments with D(2)O and d(2-)formic acid as eluent was used to determine which conformation is involved in each peak.
authors: Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique, Université Paul Sabatier, Toulouse, France.
authors: Marlène
authors: M
authors: Lacroix
authors: Jean-Christophe
authors: JC
authors: Garrigues
authors: François
authors: F
authors: Couderc
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003198:Computer Simulation; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004745:Enkephalins / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D009281:Naphthalenes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D004745:Enkephalins; D009281:Naphthalenes; C027717:2,3-naphthalenedicarboxaldehyde
references: J Chromatogr A. 1998 Jan 23;794(1-2):85-96
references: 9491558
references: Biopolymers. 2004 Aug 15;74(6):476-94
references: 15274091
references: J Neurosci Methods. 2006 Jan 15;150(1):74-9
references: 16051372
references: J Pharm Biomed Anal. 1990;8(5):419-29
references: 2081203
references: Med Sci Monit. 2005 May;11(5):MS47-53
references: 15874900
references: J Mass Spectrom. 2005 Feb;40(2):146-53
references: 15706617
references: Neuropeptides. 2002 Aug;36(4):281-6
references: 12372702
references: J Sep Sci. 2005 Dec;28(18):2534-9
references: 16405185
references: Biophys J. 2004 Mar;86(3):1587-600
references: 14990485
references: Electrophoresis. 2005 Jun;26(13):2608-21
references: 15948219
references: Rapid Commun Mass Spectrom. 2005;19(1):1-8
references: 15568184
references: Nature. 1975 Dec 18;258(5536):577-80
references: 1207728
references: Rapid Commun Mass Spectrom. 2001;15(23):2341-6
references: 11746901
references: J Am Chem Soc. 2002 Jun 5;124(22):6398-406
references: 12033871
references: J Mass Spectrom. 2003 Nov;38(11):1178-86
references: 14648825
references: J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Nov 25;780(2):269-81
references: 12401352
references: Neurosci Lett. 1993 Oct 14;161(1):77-80
references: 8255552
references: FEBS Lett. 1987 Mar 23;213(2):265-8
references: 3030814
references: J Sep Sci. 2005 Sep;28(14):1779-84
references: 16224973
references: J Pharm Biomed Anal. 1992 Jan;10(1):49-60
references: 1391083
references: J Chromatogr. 1989 Nov 10;496(1):55-70
references: 2592517
references: J Chromatogr B Biomed Appl. 1996 May 31;681(1):69-76
references: 8798914