Identification of Peptides and Proteins in Illegally Distributed Products by MALDI-TOF-MS

An analytical strategy based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for identification of peptides and proteins in illegally distributed products is presented. The identified compounds include human growth hormone (hGH), human somatoliberin, anti-obesity drug (AOD), growth hormone releasing peptides (GHRP-2 and GHRP-6), Glycine-GHRP-2 and Glycine-GHRP-6, ipamorelin, insulin aspart and porcine, delta sleep-inducing peptide (DSIP), thymosin β4, insulin like growth factor (IGF), mechano growth factor (MGF), human chorionic gonadotropin (hCG), melanotan II, bremelanotide, dermorphin and body protecting compound (BPC 157). The identification of proteins was mainly based on peptide mass fingerprinting, i.e., bottom up approach, while the smaller peptides were identified through de-novo sequencing. In cases when a reference standard was available, complementary identification was performed by capillary electrophoresis in double-injection mode (DICE), where a suspicious product was compared with the reference standard through two consecutive injections within the same electrophoretic run

Separation of Pharmaceuticals by Capillary Electrophoresis using Partial Filling and Multiple-injections

Different multiple-injection methodologies and the partial filling technique (PFT) have been utilized for separation of pharmaceuticals by capillary elec-trophoresis. In multiple-injection capillary zone electrophoresis (MICZE), the samples and all standards, used for construction of the calibration curve, are analyzed within a single run. Four different modes of MICZE have been described by means of equations, which were experimentally verified. The developed equations facilitate the transfer from conventional single-injection CZE to one or more of these MICZE-modes, depending on the selectivity between the analyte and the injection marker. The applicability of two of these modes was then demonstrated by quantification of buserelin and salbutamol, re-spectively in commercially available pharmaceutical products. The content of buserelin in an injection solution was determined to 0.94 mg/ml, which only deviated slightly from the declared concentration (1 mg/ml). An alter-native mode of MICZE, offering a higher number of sequential sample injec-tions, was then utilized for single-run determination of salbutamol in 15 tab-lets, with a labelled content of 8 mg. The average content of the tablets was determined to 7.8 mg, with an intra-tablet variation of 3 % or less. Moreover, UV- and mass-spectrometric detection of enantiomeric amines, resolved by non-aqueous capillary electrophoresis (NACE), was demon-strated. Separation of enantiomeric amines was achieved using the chiral selector (-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid, (-)-DIKGA. Introduction of the non-volatile (-)-DIKGA into the mass-spectrometer was avoided by using the PFT, where the capillary is only partially filled with electrolyte containing the chiral selector

Separation of Pharmaceuticals by Capillary Electrophoresis using Partial Filling and Multiple-injections

Different multiple-injection methodologies and the partial filling technique (PFT) have been utilized for separation of pharmaceuticals by capillary elec-trophoresis. In multiple-injection capillary zone electrophoresis (MICZE), the samples and all standards, used for construction of the calibration curve, are analyzed within a single run. Four different modes of MICZE have been described by means of equations, which were experimentally verified. The developed equations facilitate the transfer from conventional single-injection CZE to one or more of these MICZE-modes, depending on the selectivity between the analyte and the injection marker. The applicability of two of these modes was then demonstrated by quantification of buserelin and salbutamol, re-spectively in commercially available pharmaceutical products. The content of buserelin in an injection solution was determined to 0.94 mg/ml, which only deviated slightly from the declared concentration (1 mg/ml). An alter-native mode of MICZE, offering a higher number of sequential sample injec-tions, was then utilized for single-run determination of salbutamol in 15 tab-lets, with a labelled content of 8 mg. The average content of the tablets was determined to 7.8 mg, with an intra-tablet variation of 3 % or less. Moreover, UV- and mass-spectrometric detection of enantiomeric amines, resolved by non-aqueous capillary electrophoresis (NACE), was demon-strated. Separation of enantiomeric amines was achieved using the chiral selector (-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid, (-)-DIKGA. Introduction of the non-volatile (-)-DIKGA into the mass-spectrometer was avoided by using the PFT, where the capillary is only partially filled with electrolyte containing the chiral selector

Peptide ion channel toxins from the bootlace worm, the longest animal on Earth

Polypeptides from animal venoms have found important uses as drugs, pharmacological tools, and within biotechnological and agricultural applications. We here report a novel family of cystine knot peptides from nemertean worms, with potent activity on voltage-gated sodium channels. These toxins, named the alpha-nemertides, were discovered in the epidermal mucus of Lineus longissimus, the 'bootlace worm' known as the longest animal on earth. The most abundant peptide, the 31-residue long alpha-1, was isolated, synthesized, and its 3D NMR structure determined. Transcriptome analysis including 17 species revealed eight alpha-nemertides, mainly distributed in the genus Lineus. alpha-1 caused paralysis and death in green crabs (Carcinus maenas) at 1 mu g/kg (similar to 300 pmol/kg). It showed profound effect on invertebrate voltage-gated sodium channels (e.g. Blattella germanica Na(v)1) at low nanomolar concentrations. Strong selectivity for insect over human sodium channels indicates that a-nemertides can be promising candidates for development of bioinsecticidal agents