From the culture broth of the mould Trichoderma viride, strain NRRL 3199, a microheterogeneous mixture of the membrane active 20-residue peptaibol alamethicin (ALM) could be isolated. ALMs were isolated by XAD-2 column chromatography and separated by silica gel chromatography and trichloromethane/MeOH gradient elution into an acidic and neutral group of peptides, named ALM F30 and ALM F50, respectively, according to their 100 Rf on TLC. Peptides ALM F50 were separated by semi-preparative and analytical HPLC and subjected to ESI-MS. Ten sequences of ALM F30 and their relative quantities could be determined. The major peptides ALM F30/3 (46%) and ALM F30/7 (40%), distinguished by Aib/Ala exchange in position 6, correspond to sequences described as ALM I and II occurring in the original alamethicin from Upjohn Company. Analogously, 13 sequences of the neutral peptide mixture named ALM F50 could be determined. The major peptide ALM F50/5 (75%) and the minor peptide ALM F50/7 (10%) are distinguished from ALM F30/3 and ALM F30/7 by having Gln17 in place of Glu17, the latter occurring in the F30 group. Notably. currently commercially available alamethicins (Fluka, Sigma) represent microheterogeneous mixtures of the neutral ALM F50 peptides with trace amounts of acidic ALM F30 peptides.

"Proteomics" and "peptidomics" are used as technical terms to define the analysis and study of all proteins and peptides expressed in an organism or tissue. In analogy we propose the name peptaibiomics for the analysis of a group of fungal peptide antibiotics (peptaibiotics) containing the characteristic amino acid Aib (alpha-aminoisobutyric acid). In analogy to the peptidome the complete expression of peptaibiotics by fungal multienzyme complexes should be named the peptaibiome. Peptaibiotics are defined as peptides containing Aib and exerting a variety of bioactivities. They comprise the sub-groups of N-acetylated peptaibols, characterized also by a C-terminal amide-linked 2-amino alcohol, and lipopeptaibols having in place of an acetyl group a lipophilic fatty acid acyl group. Furthermore, lipoaminopeptides are also known with long-chain fatty acid on the N-termini, a lipoamino acid in position three and a strongly basic secondary or tertiary amine form a subgroup of mixed forms which could not be integrated in one of these three previously mentioned groups. Here we present a specific and rapid screening method on the peptaibiome applicable directly onto filamentous fungi cultured in a single Petri dish. The method comprises solid-phase extraction (SPE) of peptaibiotics followed by on-line reversed-phase HPLC coupled to an ion trap electrospray tandem mass spectrometer (ES-MS). The presence of these peptides is indicated by characteristic mass differences of Deltam = 85.1 Da representing Aib-residues which can be observed in the b-series of acylium fragment ions resulting from ES-MS. Partial sequences can be deduced from the data and compared with structures compiled in electronic peptaibol data bases. The judgement is possible whether or not structures are novel, already known or related to known structures. Suitability of the method is demonstrated with the analysis of strains of Trichoderma and its teleomorph Hypocrea. New sequences of peptaibiotics are presented and those being related to established 10- to 18-residue peptaibols trichovirin, trichogin and trichotoxin, which have been described in the literature.

From the culture broth of the mold Trichoderma viride, strain 63 C-I, the polypeptide antibiotic suzukacillin (SZ) was isolated. A peptide mixture named SZ-A was obtained by crystallization from crude SZ. Individual peptides from SZ-A were isolated by semipreparative HPLC and sequences were determined by HPLC-ESI-MS. The data confirm a general sequence of SZ-A published previously and in addition establish the individual sequences of 15 acetylated eicosa peptides with C-terminal alcohols. The major peptide SZ-A4 (21% of all peptides) shows the sequence:Ac-Aib-Ala-Aib-Ala-Aib-Ala(6)-Gln-Aib-Lx(9)-Aib-Gly-Aib(12)-Aib-Pro-Vx(15)-Aib-Vx(17)-Gln-Gln-Fol. Amino acid exchanges of the peptaibol are located in position 6 (Ala/Aib), 9 (Vx/Lx), 12 (Aib/Lx), 17 (Aib/Vx) and possibly at position15 (Val/Iva) (uncommon abbreviations: Aib (alpha-aminoisobutyric acid); Iva (D-isovaline); Lx (L-leucine or L-isoleucine); Vx (L-valine or D-isovaline); Fol (L-phenylalaninol)).

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From the culture broth of the filamentous fungus Trichoderma parceramosum, strain CBS 936.69, a mixture of polypeptide antibiotics (pepaibiotics), named trichobrachin (TB), was isolated. Three major groups designated TB I, TB II, and TB III could be separated and isolated by preparative TLC on silica gel. Individual peptides of these three groups were sequenced by on-line LC/ESI-MS(n). The mixture of N-acetylated peptides comprises ten 19-residue peptides with a free C-terminal Gln residue (TB I peptides), two 18-residue peptides with a free C-terminal Gln residue (TB II 1 and 2), seven 20-residue peptides with a C-terminal amide-bound phenylalaninol (TB II 3-10), and 34 eleven-residue peptides with either a C-terminal leucinol or isoleucinol or valinol (TB III 1-34). Monitoring production and degradation of peptaibiotics in a pilot experiment revealed that the biosynthesis of TB II and TB III peptides starts two days after the beginning of fermentation. After five days of fermentation, the concentration of TB II decreased, whereas the amount of TB I increased. This observation unequivocally demonstrates that those two 18-residue TB I and TB II peptides with the free carboxy terminus result from enzymatic C-terminal degradation of the 20-residue TB II peptides. In analogy to the technical terms proteome and proteomics, the terms peptaibiome and peptaibiomics have recently been proposed for the entirety and dynamics of the Aib-containing peptides (comprehensively named peptaibiotics). Consequently, the entire peptaibiome of T. parceramosum grown under submerse conditions in shake-flasks for five days comprises at least 54 peptides differing in main-chain length and microheterogeneity, i.e., exchange of amino acids and the C-terminal 1,2-amino alcohol.