Determination of disulfide linkages in antimicrobial peptides of the macin family by combination of top-down and bottom-up proteomics

UNLABELLED: Macins are a distinct class of antimicrobial peptides (AMPs) produced by leeches and Hydra. Their function depends strongly on their three-dimensional structure. In order to support structural elucidation of these AMPs, the knowledge and proper assignment of disulfide bonds formed in these cysteine-rich peptides is a prerequisite. In this report, we outline an analytical strategy, encompassing a combination of top-down MS based analytics and sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution MS/MS analysis by LTQ-Orbitrap MS to assign disulfide linkages of three members of the macin family, namely neuromacin, theromacin, and hydramacin-1. The results revealed that the eight cysteine residues conserved in all three macins form the same four disulfide bonds, i.e. [C1:C6], [C2:C5], [C3:C7], and [C4:C8]. Theromacin, which possess two additional cysteine residues, forms a fifth disulfide bond. BIOLOGICAL SIGNIFICANCE: Beside the high biological significance which is based on the inherent dependence of biological activity on the structural features of antimicrobial peptides (which holds true for entirely every protein), the presented analytical strategy will be of wide interest, as it widens the available toolbox for the analysis of this important posttranslational modification.

Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.

Combined top-down and bottom-up mass spectrometry resolves the disulfide-bond architecture of leech-derived macin family antimicrobial peptides neuromacin, theromacin, and hydramacin-1, anchoring their structure-function relationships.

Leech-derived molecules this paper discusses, each with a full profile in the ASH compound registry:

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