Single-pot, Solid-Phase-enhanced Sample Preparation for Proteomics Experiments

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.