Open AccessSolid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics
Author(s) -
Cecil J. Howard,
Brendan M. Floyd,
Angela M. Bardo,
Jagannath Swaminathan,
Edward M. Marcotte,
Eric V. Anslyn
Publication year - 2020
Publication title -
acs chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.899
H-Index - 111
eISSN - 1554-8937
pISSN - 1554-8929
DOI - 10.1021/acschembio.0c00040
Subject(s) - derivatization , peptide , chemistry , proteolysis , proteomics , proteome , combinatorial chemistry , lysis , molecule , chromatography , reagent , quantitative proteomics , small molecule , bottom up proteomics , biochemistry , mass spectrometry , organic chemistry , tandem mass spectrometry , enzyme , gene , protein mass spectrometry
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.