ABSTRACT
The multicomponent venom of the spider Cupiennius salei was separated by three different chromatographic strategies to facilitate subsequent analysis of peptidic venom components by tandem mass spectrometry (MALDI-TOF-MS and ESI-MS), Edman degradation and amino acid analysis: (a) desalting of the crude venom by RP-HPLC only, (b) chromatographic separation of the crude venom into 42 fractions by RP-HPLC, and (c) multidimensional purification of the crude venom by size exclusion and cation exchange chromatography and RP-HPLC. A total of 286 components were identified in the venom of C. salei by mass spectrometry and the sequence of 49 new peptides was determined de novo by Edman degradation and tandem mass spectrometry; 30 were C-terminally amidated. The novel peptides were assigned to two main groups: (a) short cationic peptides and (b) Cys-containing peptides with the inhibitor cystine knot motif. Bioinformatics revealed a limited number of substantial similarities, namely with the peptides CpTx1 from the spider Cheiracantium punctorium and U3-ctenitoxin-Asp1a from the South American fishing spider (Ancylometes sp.) and with sequences from a Lycosa singoriensis venom gland transcriptome analysis. The results clearly indicate that the quality of the data is strongly dependent on the chosen separation strategy. The combination of orthogonal analytical methods efficiently excludes alkali ion and matrix adducts, provides indispensable information for an unambiguous identification of isomasses, and results in the most comprehensive repertoire of peptides identified in the venom of C. salei so far.
Subject(s)
Spider Venoms/chemistry , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Computational Biology , Molecular Sequence Data , Peptides/chemistry , Peptides/genetics , Peptides/isolation & purification , Sequence Alignment , Sequence Homology, Amino Acid , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spider Venoms/genetics , Spider Venoms/isolation & purification , Spiders/chemistry , Spiders/genetics , Tandem Mass SpectrometryABSTRACT
Two isomeric dialkynylpyrene phosphoramidites and their incorporation into oligonucleotides are described. The pyrene units closely resemble the well-known perylene bisimide dye PDI with regard to the ability to self-organize within a DNA duplex. In addition, dialkynylpyrenes exhibit significant monomer and remarkably strong excimer fluorescence. The dialkynylpyrene building blocks are promising candidates for applications in diagnostic tools, such as excimer-based molecular beacons, as well as for novel DNA-based materials with special optical properties.
