A critical step in proteomics analysis is the optimal extraction and processing of protein material to ensure the highest sensitivity in downstream detection. Achieving this requires a sample-handling technology that exhibits unbiased protein manipulation, flexibility in reagent use, and virtually lossless processing. Addressing these needs, the single-pot, solid-phase-enhanced sample-preparation (SP3) technology is a paramagnetic bead-based approach for rapid, robust, and efficient processing of protein samples for proteomic analysis. SP3 uses a hydrophilic interaction mechanism for exchange or removal of components that are commonly used to facilitate cell or tissue lysis, protein solubilization, and enzymatic digestion (e.g., detergents, chaotropes, salts, buffers, acids, and solvents) before downstream proteomic analysis. The SP3 protocol consists of nonselective protein binding and rinsing steps that are enabled through the use of ethanol-driven solvation capture on the surface of hydrophilic beads, and elution of purified material in aqueous conditions. In contrast to alternative approaches, SP3 combines compatibility with a substantial collection of solution additives with virtually lossless and unbiased recovery of proteins independent of input quantity, all in a simplified single-tube protocol. The SP3 protocol is simple and efficient, and can be easily completed by a standard user in ~30 min, including reagent preparation. As a result of these properties, SP3 has successfully been used to facilitate examination of a broad range of sample types spanning simple and complex protein mixtures in large and very small amounts, across numerous organisms. This work describes the steps and extensive considerations involved in performing SP3 in bottom-up proteomics, using a simplified protein cleanup scenario for illustration.

中文翻译：

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单锅固相增强样品制备，用于蛋白质组学实验。

蛋白质组学分析的关键步骤是蛋白质材料的最佳提取和加工，以确保下游检测的最高灵敏度。为此，需要一种样品处理技术，该技术应显示出无偏的蛋白质操作，试剂使用的灵活性以及几乎无损的处理方法。为了满足这些需求，单罐固相增强样品制备（SP3）技术是一种基于顺磁微珠的方法，可以快速，稳健和有效地处理蛋白质样品以进行蛋白质组学分析。SP3使用亲水相互作用机制来交换或去除通常用于促进细胞或组织裂解，蛋白质溶解和酶消化（例如去污剂，离液剂，盐，缓冲液，酸和溶剂）的组分（在下游蛋白质组分析之前）。SP3协议包括非选择性蛋白质结合和漂洗步骤，这些步骤可通过使用亲水性珠粒表面上乙醇驱动的溶剂化捕获以及在水性条件下洗脱纯化的物质来实现。与替代方法相反，SP3将兼容性与大量溶液添加剂相结合，并且在无输入量的情况下几乎无损失且无偏见地回收了蛋白质，所有这些均以简化的单管操作规程进行。SP3协议简单有效，可以由标准用户在大约30分钟内轻松完成，包括试剂制备。由于这些特性，SP3已成功用于促进多种样品类型的检查，这些样品类型涵盖了简单和复杂蛋白质混合物的大量和非常少量，涵盖了多种生物。