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Micro-size exclusion chromatography coupled with capillary liquid chromatography (capLC) and mass spectrometry (MS) provides a rapid and simple approach to the preliminary screening of active ligands toward a specific target macromolecule. In this study, the effectiveness of this technique is demonstrated by a number of small molecule ligands with known binding affinities towards the protein target. All ligands were incubated together with a target protein under native conditions. Separation was then achieved by microcentrifugation where the high molecular weight (MW) compounds were selectively passed through the size-exclusion material. The retained low MW compounds were then recovered and analyzed by capLC/MS. The absence of the ligand indicated strong affinity towards the target, while ligand detection indicated inactivity. This assay demonstrated the drugs that were acting as strong inhibitors of Co-PDF from those showing to be comparatively inactive. The relative binding rank order of the drugs towards Co-PDF was also determined. The results were validated by a corresponding set of control experiments in which the target molecules were excluded from the process. In principle, high-throughput micro-size exclusion chromatography, coupled with capLC/MS, offers a powerful technique as a preliminary screen in determining both the strong binding affinity and the relative affinity rank ordering of ligands towards a specific target macromolecule, and is complementary with other analytical drug screening techniques., (Copyright 2001 John Wiley & Sons, Ltd.)

MS/MS data derived from the [M-H](-) ions of desulfated caerulein peptides provide (i) sequencing information from a combination of alpha, beta and gamma backbone cleavages, and (ii) identification of specific amino acid side chains by side-chain cleavages [e.g. Ser (-CH(2)O), Thr (-CH(3)CHO) and Asp (-H(2)O)] (fragmentations having no counterparts in positive ion spectra). In addition, delta and/or gamma backbone cleavage ions from Asp residues identify the position of these residues in the peptide. In contrast, neither delta nor gamma cleavage ions are observed from either the Gln2 residue nor from Phe residues. Full structural information can be obtained from a consideration of the positive and negative ion MS/MS data in concert., (Copyright 2002 John Wiley & Sons, Ltd.)

Sixteen caerulein-type peptides have been isolated from the skin secretions of the Australian Blue Mountains tree frog Litoria citropa. There are four groups of these peptides. The first is based on the structure of the known neuropeptide caerulein [pEQDY(SO(3))TGWMDF-NH(2)], now renamed caerulein 1.1. Examples of peptides of the other groups are as follows: caerulein 2.1 [pEQDY(SO(3))TGAHMDF-NH(2)], caerulein 3.1 [pEQDY(SO(3))GTGWMDF-NH(2)] and caerulein 4.1 [pEQDY(SO(3))TGSHMDF-NH(2)]. All of these peptides are accompanied by the associated peptide where Phe replaces Met, and all eight of the caerulein peptides are accompanied by the desulfated analogues. Negative ion electrospray mass spectrometry (ES-MS) is used to determine the molecular weights of the caeruleins 1-4 [from their [M - H](-) ions], while the sequences of the peptides are determined from the B and Y + 2 cleavage ions in the mass spectra of the [MH(+) - SO(3)](+) ions., (Copyright 1999 John Wiley & Sons, Ltd.)

A combination of electrospray mass spectrometry, Lys-C digest/mass spectrometry and automated Edman sequencing provides the amino acid sequences of nineteen citropin peptides isolated from the granular dorsal and submental glands of the Blue Mountains tree frog Litoria citropa. Citropin 1.1 [Gly Leu Phe Asp Val Ile Lys Lys Val Ala Ser Val Ile Gly Gly Leu (NH(2))] and citropin 1.2 [Gly Leu Phe Asp Ile Ile Lys Lys Val Ala Ser Val Val Gly Gly Leu (NH(2))] are the two major skin peptides: both show significant wide-spectrum antibacterial activity., (Copyright 1999 John Wiley & Sons, Ltd.)

Electrospray mass spectrometry and automated Edman sequencing provides the structures of two new caerin 1 antimicrobial peptides from the skin glands of the Australian tree frog Litoria chloris. These are: caerin 1.8 Gly Leu Phe Lys Val Leu Gly Ser Val Ala Lys His Leu Leu Pro His Val Val Pro Val Ile Ala Glu Lys Leu (NH2), and caerin 1.9, Gly Leu Phe Gly Val Leu Gly Ser Ile Ala Lys His Val Leu Pro His Val Val Pro Val Ile Ala Glu Lys Leu (NH2).

The glandular secretions of the skin of Litoria rubella specimens collected from five locations on the eastern seaboard of Queensland (Australia) contain the three tryptophyllin peptides Phe Pro Trp Leu (NH2), Phe Pro Trp Pro (NH2) and Phe Pro Phe Pro Trp Leu (NH2). The relative proportions of these peptides in the glandular secretion are associated with geographic location, i.e. Phe Pro Trp Pro (NH2) is a minor component of the peptide mixture in frogs from southern Queensland, but becomes significantly more abundant as the location becomes more northerly. This trend indicates an evolutionary change in the animal, but for what reason, and over what timescale is not known at this time.