Successful proteomic characterization of biological material depends on the development of robust sample processing methods. The acorn barnacle Amphibalanus amphitrite is a biofouling model for adhesive processes, but the identification of causative proteins involved has been hindered by their insoluble nature. Although effective, existing sample processing methods are labor and time intensive, slowing progress in this field. Here, a more efficient sample processing method is described which exploits pressure cycling technology (PCT) in combination with protein solvents. PCT aids in protein extraction and digestion for proteomics analysis. Barnacle adhesive proteins can be extracted and digested in the same tube using PCT, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. PCT methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than highly polar hexafluoroisopropanol, leading to the identification and description of >40 novel proteins at the interface. Some of these have homology to proteins with elastomeric properties or domains involved with protein-protein interactions, while many have no sequence similarity to proteins in publicly available databases, highlighting the unique adherent processes evolved by barnacles. The methods described here can not only be used to further characterize barnacle adhesive to combat fouling, but may also be applied to other recalcitrant biological samples, including aggregative or fibrillar protein matrices produced during disease, where a lack of efficient sample processing methods has impeded advancement. Data are available via ProteomeXchange with identifier PXD012730.

中文翻译：

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挑战蛋白质组学样品处理的压力循环技术：在藤壶粘合剂中的应用

生物材料的成功蛋白质组学表征取决于强大的样品处理方法的发展。橡子藤壶Amphibalanus amphitrite是用于粘附过程的生物污染模型，但是所涉及的致病蛋白的鉴定由于其不溶性而受到阻碍。尽管有效，但是现有的样品处理方法是费时费力的，这减慢了该领域的进展。在此，描述了一种更有效的样品处理方法，该方法利用了压力循环技术（PCT）与蛋白质溶剂的结合。PCT有助于蛋白质组学分析的蛋白质提取和消化。藤壶黏附蛋白可以使用PCT在同一试管中提取和消化，从而最大程度地减少样品损失，将通量增加到16个同时处理的样品，并将样品处理时间减少到8小时以下。与以前的方法相比，PCT方法产生了相似的蛋白质组。与高极性六氟异丙醇相比，两种在环境压力下不能有效地从胶粘剂中提取蛋白质的溶剂（尿素和甲醇）比在高极性六氟异丙醇中产生更多的蛋白质鉴定，从而导致在界面上鉴定和描述了40多种新型蛋白质。其中一些与具有弹性性质的蛋白质或涉及蛋白质-蛋白质相互作用的结构域具有同源性，而许多与公开数据库中的蛋白质没有序列相似性，突出了藤壶进化出的独特粘附过程。本文描述的方法不仅可以用于进一步表征藤壶粘胶以防止结垢，而且还可以应用于其他顽固的生物学样品，包括疾病期间产生的聚集性或纤维状蛋白基质，缺乏有效的样品处理方法阻碍了其发展。数据可通过ProteomeXchange获得，其标识符为PXD012730。