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bioRxiv · 10.1101/2020.01.13.904540

Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics

Howard, C. J.·Floyd, B. M.·Bardo, A. M.·Swaminathan, J.·Marcotte, E. M.·Anslyn, E. V.
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Abstract

The field of proteomics has expanded recently with more sensitive techniques for the bulk measurement of peptides as well as single-molecule techniques. One limiting factor for some of these methods is the need for multiple chemical derivatizations and highly pure proteins free of contaminants. We demonstrate a solid-phase capture strategy suitable for the proteolysis, purification, and subsequent chemical modification of peptides. We use this resin on an HEK293T cell lysate and perform one-pot proteolysis, capture, and derivatization to generate a cellular proteome that identified over 40,000 bead-bound peptides. We also show that this capture can be reversed in a traceless manner, such that it is amenable for single-molecule proteomics techniques. With this technique, we perform a fluorescent labeling and C-terminal derivatization on a peptide and subject it to fluorosequencing, demonstrating that washing the resin is sufficient to remove excess dyes and other reagents prior to single-molecule protein sequencing.

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Howard, C. J., Floyd, B. M., Bardo, A. M., Swaminathan, J., Marcotte, E. M., Anslyn, E. V.. 2020-01-14. Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics. https://doi.org/10.1101/2020.01.13.904540

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