Quantitative proteomics via mass spectrometry can provide valuable insight into molecular and phenotypic characteristics of a living system. Recent mass spectrometry developments include data‐independent acquisition (SWATH/DIA‐MS), an accurate, sensitive and reproducible method for analysing the whole proteome. The main requirement for this method is the creation of a comprehensive spectral library. New technologies have emerged producing larger and more accurate species‐specific libraries leading to a progressive collection of proteome references for multiple molecular model species. Here, for the first time, we set out to compare different spectral library constructions using multiple tissues from a coral reef fish to demonstrate its value and feasibility for nonmodel organisms. We created a large spectral library composed of 12,553 protein groups from liver and brain tissues. Via identification of differentially expressed proteins under fish exposure to elevated pCO₂ and temperature, we validated the application and usefulness of these different spectral libraries. Successful identification of significant differentially expressed proteins from different environmental exposures occurred using the library with a combination of data‐independent and data‐dependent acquisition methods as well as both tissue types. Further analysis revealed expected patterns of significantly up‐regulated heat shock proteins in a dual condition of ocean warming and acidification indicating the biological accuracy and relevance of the method. This study provides the first reference spectral library for a nonmodel organism. It represents a useful guide for future building of accurate spectral library references in nonmodel organisms allowing the discovery of ecologically relevant changes in the proteome.

中文翻译：

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探索 SWATH-MS 作为非模型鱼蛋白质组水平量化的工具。

通过质谱的定量蛋白质组学可以提供对生命系统的分子和表型特征的宝贵见解。最近的质谱发展包括数据独立采集 (SWATH/DIA-MS)，这是一种用于分析整个蛋白质组的准确、灵敏和可重复的方法。这种方法的主要要求是创建一个全面的光谱库。新技术的出现产生了更大、更准确的物种特异性文库，从而逐步收集多个分子模型物种的蛋白质组参考。在这里，我们首次开始使用珊瑚礁鱼的多种组织来比较不同的光谱库构建，以证明其对非模式生物的价值和可行性。我们创建了一个由 12 个组成的大型光谱库，来自肝脏和脑组织的 553 个蛋白质组。通过鉴定鱼暴露于升高的 pCO 下差异表达的蛋白质₂和温度，我们验证了这些不同光谱库的应用和有用性。使用该文库结合数据独立和数据依赖的采集方法以及两种组织类型，成功鉴定了来自不同环境暴露的显着差异表达的蛋白质。进一步的分析揭示了在海洋变暖和酸化的双重条件下热休克蛋白显着上调的预期模式，表明该方法的生物学准确性和相关性。这项研究为非模式生物提供了第一个参考光谱库。它代表了未来在非模式生物中建立准确的光谱库参考的有用指南，允许发现蛋白质组中与生态相关的变化。