title: Investigating of labelling and detection of transthyretin synthetic peptide derivatized with naphthalene-2,3-dicarboxaldehyde.
abstract: Labelling and detection of a synthetic peptide (PN) mimicking a tryptic fragment of interest for the diagnosis of familial amyloidal polyneuropathy have been investigated optically and electrochemically. We decided to covalently label naphtalene-2,3-dicarboxyaldehyde (NDA), a fluorogenic and electroactive molecule on PN. First, the optimization of the labelling chemical reaction was performed by capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF). The analytical parameters such as separation efficiency and peak area were considered to propose this optimized derivatization reaction. The results obtained allowed us to establish the pH and ionic strength of the derivatization buffer, the molar ratio between NDA and PN and the reaction time of the labelling. Optimal conditions are obtained when [NDA]/[PN]=40, buffer pH of 9, buffer ionic strength of 70 mM and reaction time of 15 min. Second, differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were also used to characterize NDA-labelled PN and different electroinactive amino acids (histidine, lysine, serine, threonine) which are in the PN sequence. The electrochemical detection experiments demonstrated that the labelled biomolecules could be also easily detected at low concentration. Moreover, the derivatization reaction could be followed to describe more precisely the labelling process of these biomolecules. Optimal conditions for labelling are obtained when [NDA]total/[CN(-)] ratio =1 and [NDA]total/[amino acid or peptide]=100 with a buffer having a pH=9 on a glassy carbon electrode. In all cases, an obvious oxidation peak for the N-2-substituted-1-cyanobenz-[f]-isoindole derivative (CBI) has been observed at 0.5-0.7 V/SCE. The multi-labelling of PN and lysine were shown with DPV. We presumed this result to occur because of the shouldered shape of the DPV peak shape. These experiments confirm that NDA can be used as a derivative agent for PN, allowing for electrochemical and fluorescence detections with a limit of detection of labelled PN estimated at 0.2 µM and 5 µM, respectively.
authors.affiliation: CNRS, Laboratoire Interfaces et Systèmes Electrochimiques, UPR 15, F-75252 Paris, France; UPMC, LISE UPR 15 CNRS; 4 Place Jussieu, case postale 133, F-75252 Paris, France.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Faure
authors.forename: Sonia
authors.initials: S
authors.lastname: Korchane
authors.forename: Isabelle
authors.initials: I
authors.lastname: Le Potier
authors.forename: Antoine
authors.initials: A
authors.lastname: Pallandre
authors.forename: Claude
authors.initials: C
authors.lastname: Deslouis
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Haghiri-Gosnet
authors.forename: Jean
authors.initials: J
authors.lastname: Gamby
mesh_terms: D000595:Amino Acid Sequence; D028227:Amyloid Neuropathies, Familial / Q000175:diagnosis; D002244:Carbon / Q000737:chemistry; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D019075:Electrophoresis, Capillary; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000737:chemistry*; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D011228:Prealbumin / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
chemical_list: D009281:Naphthalenes; D010455:Peptides; D011228:Prealbumin; D012996:Solutions; C027717:2,3-naphthalenedicarboxaldehyde; D002244:Carbon; D014357:Trypsin
keywords: Capillary electrophoresis; Differential pulse voltammetry; Naphthalene-2,3-dicarboxaldehyde; Transthyretin