Fragmentation Follows Structure: Top-Down Mass Spectrometry Elucidates the Topology of Engineered Cystine-Knot Miniproteins

Over the last decades the field of pharmaceutically relevant peptides has enormously expanded. Among them, several peptide families exist that contain three or more disulfide bonds. In this context, elucidation of the disulfide patterns is extremely important as these motifs are often prerequisites for folding, stability, and activity. An example of this structure-determining pattern is a cystine knot which comprises three constrained disulfide bonds and represents a core element in a vast number of mechanically interlocked peptidic structures possessing different biological activities. Herein, we present our studies on disulfide pattern determination and structure elucidation of cystine-knot miniproteins derived from Momordica cochinchinensis peptide MCoTI-II, which act as potent inhibitors of human matriptase-1. A top-down mass spectrometric analysis of the oxidised and bioactive peptides is described. Following the detailed sequencing of the peptide backbone, interpretation of the MS3 spectra allowed for the verification of the knotted topology of the examined miniproteins. Moreover, we found that the fragmentation pattern depends on the knottin’s folding state, hence, tertiary structure, which to our knowledge has not been described for a top-down MS approach before.近数十年来，药用相关肽领域得到了长足发展。其中，存在多个含有三对及以上二硫键的肽家族。在此背景下，二硫键配对模式（disulfide patterns）的解析至关重要，因为这类结构基序往往是肽链折叠、稳定性及生物活性的必要前提。 这类决定肽链结构的模式的典型示例之一便是胱氨酸结（cystine knot）：其包含三对受约束的二硫键，是大量具备不同生物活性的机械互锁肽类结构的核心组成单元。 本文中，我们针对源自木鳖（Momordica cochinchinensis）肽MCoTI-II的胱氨酸结微型蛋白开展了二硫键配对模式解析与结构阐释研究，这类蛋白是人类膜型丝氨酸蛋白酶matriptase-1的强效抑制剂。本研究阐述了针对氧化态活性肽的自上而下质谱法（top-down mass spectrometry）分析方法。在完成肽链骨架的详细测序后，通过解析三级质谱（MS3）谱图，我们验证了所研究微型蛋白的打结拓扑结构。 此外，我们发现结蛋白（knottin）的碎裂模式取决于其折叠状态，即三级结构；据我们所知，这一现象此前尚未在自上而下质谱法的相关研究中被报道过。