MassIVE MSV000087551

Complete Public PXD026393

Photoredox-catalyzed decarboxylative C-terminal differentiation for bulk and single molecule proteomics

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Description

Methods for the selective labeling of functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. Photoredox methods in discriminative targeting of the terminal carboxylic acid from the internal acidic residues have been described but not effectively applied in concert with peptide-centric proteomic technologies. In this work, we describe methods for the use of lumiflavin based photoredox chemistry in peptide mass-spectrometry and single molecule protein sequencing. After optimizing the instrumentation and reaction conditions with the peptide angiotensin, we demonstrate successfully labeling of peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts. Using two distinct analytical approaches, we characterize labeling efficiencies of greater than 50% across full proteomes and determine biases due to different C-terminal amino acids, finding histidine to be preferentially disfavored. Finally we demonstrate the ability to fluorescently label an internal acidic residue following C-terminal carboxylic acid derivatization, as suitable for single molecule protein sequencing technologies. The strategies and limitations described in this work provide clear guidance for further improvement and general use of this chemistry for analyzing proteins. [doi:10.25345/C55R8P] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: C-terminal ; Bioconjugation

Contact

Principal Investigators:
(in alphabetical order) 		Edward Marcotte, University of Texas-Austin, United States
Submitting User: bfloyd

Publications

Le Zhang, Brendan M Floyd, Maheshwerreddy Chilamari, James Mapes, Jagannath Swaminathan, Steven Bloom, Edward M Marcotte, Eric V Anslyn.
Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics.
ACS Chem. Biol. 2021 Nov; 16, 2595-2603.

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Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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