Dataset Information

Peptide N-terminal Bead Capture and Release

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 these methods is the need for multiple chemical derivatizations and highly pure proteins free of contaminants. We show 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 8,000 proteins. 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 differentiation on a peptide and subject it to fluorosequencing demonstrating that washing the resin is sufficient to remove excess dyes prior to single-molecule protein sequencing.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryonic Stem Cell, Kidney

SUBMITTER: Brendan Floyd

LAB HEAD: Edward Marcotte

PROVIDER: PXD016291 | Pride | 2021-06-01

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	190728_HEK293T_capture_1.raw	Raw
	190728_HEK293T_capture_2.raw	Raw
	190728_HEK293T_capture_release_result.mzid.gz	Mzid
	190728_HEK293T_capture_release_result.pride.mztab.gz	Mztab
	190728_HEK293T_flowthrough_1.raw	Raw

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Publications

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

Howard Cecil J CJ Floyd Brendan M BM Bardo Angela M AM Swaminathan Jagannath J Marcotte Edward M EM Anslyn Eric V EV

ACS chemical biology 20200514 6

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 ...[more]

PMID: 32363853

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