ISSN:
0306-042X
Keywords:
Chemistry
;
Analytical Chemistry and Spectroscopy
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Chemistry and Pharmacology
Notes:
Specific and sensitive procedures have been developed which enabled the structure elucidation of the polypeptide antibiotics (peptaibols), paracelsin isolated from Trichoderma reesei, and of trichotoxin A-50 from Trichoderma viride, by fast atom bombardment and field desorption mass spectrometry. Both peptides were found to exhibit a pronounced microheterogeneity by single and multiple exchange of amino acids. Separation by analytical and semipreparative high-performance liquid chromatography (HPLC) on octadecylsilyl-bonded, reversed-phase columns afforded a series of sequence analogues for each polypeptide. Unequivocal molecular weight and sequence identifications were obtained by positive and negative ion fast atom bombardment (FAB), and field desorption (FD) mass spectrometry, in combination with a single, selective acidolytic cleavage step. Most important for the FAB mass spectral analysis of the extremely hydrophobic polypeptides was the development of oligoethylene-glycols and -glycol ethers as suitable liquid matrix systems, yielding high sensitivity and structurally significant fragment ions of high abundance. At these conditions, the positive ion FAB mass spectra exhibit regular abundant sequence ions with acylium end groups by peptide bond cleavage from the N-terminus, which provided direct sequence determinations for 13 of 20 residues of paracelsin, and for 12 of 18 residues of trichotoxin A-50. The sequence-specific fragmentation precisely continues up to, and ends at the preferential acidolytic cleavage sites (Pro residues). The remaining C-terminal sequence data was obtained by FD and FAB mass spectra of prolyl-hexa- and -heptapeptaibols produced by acidolytic cleavage in situ with aqueous trifluoroacetic acid. In contrast to the original polypeptides, glycerol as a polar matrix was most suitable for the analysis of prolylpeptaibol fragments in the hyrolysis mixture. By contrast, treatment of paracelsins and trichotoxins A-50 with trifluororacetic acid under anhydrous conditions yielded the corresponding trifluoroacetylated polypeptide derivatives which served to ascertain the position of the C-terminal amino alcohol residues. With this procedure, the structures of 14 closely related, partially isobaric sequence analogues of trichotoxin A-50, and of four sequence analogues of paracelsin were determined with amounts of material in the low nanomole range, which provide an exact pattern of the variable and the conservative sequence areas for these polypeptides.
Additional Material:
10 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bms.1200111105
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