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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.

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Fig. 1: SP3 provides a simplified platform for processing of protein samples before MS-based proteomics analysis.
Fig. 2: The behavior of SP3 beads during processing can be used to visualize protein binding and elution.
Fig. 3: SP3 generates protein samples compatible with MS analysis.
Fig. 4: SP3 generates protein samples compatible with in-depth quantitative profiling of complex mixtures.


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C.S.H. acknowledges valuable discussions with L. Radan. The authors acknowledge support from the British Columbia Cancer Foundation Multimedia team for assistance in the creation of video protocols. G.B.M. and P.H.S. acknowledge funding support from the British Columbia Cancer Foundation for this work. S.M. acknowledges funding support from the British Columbia Proteomics Network. J.K. acknowledges funding from the CellNetworks Excellence Cluster.

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Authors and Affiliations



C.S.H. and J.K. conceived the idea. C.S.H. designed the protocol, analyzed the data, and wrote the manuscript. S.M. and C.S.H. contributed to the creation of the protocol video. S.M., T.M., P.H.S., G.B.M., and J.K. contributed to editing and content of the manuscript.

Corresponding authors

Correspondence to Christopher S. Hughes or Jeroen Krijgsveld.

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The authors declare no competing interests.

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Key references using this protocol

Hughes, C. S. et al. Mol. Syst. Biol. 10, 757 (2014)

Moggridge, S., Sorensen, P. H., Morin, G. B. & Hughes, C. S. J. Proteome Res. 17, 1730–1740 (2018)

Supplementary information

Supplementary Video 1

Handling of protein samples with SP3 for MS analysis. The video depicts all steps of the SP3 protocol, along with specific visualizations to aid in interpretation of the described protocol

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Hughes, C.S., Moggridge, S., Müller, T. et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc 14, 68–85 (2019).

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