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

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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… Click to show full 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-release 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 survey peptide capture biases from over 40,000 unique peptides from a cellular proteome. 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.

Keywords: single molecule; capture release; solid phase; peptide capture; capture

Journal Title: ACS chemical biology
Year Published: 2020

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