Specific Affinity Enrichment of Electrochemically Cleaved Peptides Based on Cu(II)-Mediated Spirolactone Tagging

Abstract

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Electrochemical oxidation yielding specific cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) residues provides a potential alternative to enzymatic digestion and a possibility for further chemical labeling by introducing reactive spirolactone moieties. However, spirolactone-containing peptides suffer from low stability due to hydrolysis and intramolecular side reactions. We found that Cu(II) ions stabilize the spirolactone and prevent intramolecular side reactions during chemical labeling, allowing efficient chemical tagging with a reduced excess of labeling reagent without intramolecular side reactions. On the basis of this reaction, we developed an analytical procedure combining electrochemical digestion, Cu(II)-mediated spirolactone biotinylation, and enrichment by avidin affinity chromatography with mass spectrometry. The method was optimized with the tripeptide LWL and subsequently applied to chicken egg white lysozyme, in which one biotinylated electrochemistry (EC)-cleaved peptide was identified after affinity enrichment. This proof-of-principle shows that specific enrichment of electrochemically cleaved spirolactone-containing peptides can be used for protein identification and notably that inclusion of Cu(II) ions is essential for stabilizing spirolactones for subsequent biotinylation.

Electrochemistry-enabled residue-specific modification of peptides and proteins

“…[42][43][44][45][46][47][48][49][50][51] However, it remains underexplored as a chemical lever for the modification of peptides and proteins. [52][53][54][55][56][57][58][59][60][61][62][63] Recently, Lei's group combined different bioconjugation strategies of peptides and proteins driven by biocompatible energy input. 64 The authors nicely wrapped up different modulation protocols, employing photochemistry as well as electrochemistry as a single electron transfer (SET)-based technique.…”

Electrochemistry for the Chemoselective Modification of Peptides and Proteins

“…However, the variety of redox-active amino acids and similarities in their redox potentials complicate the task of achieving chemo­selective modification (see Figure D), particularly in the context of proteins. There has been, and continues to be, considerable effort devoted to the direct modification of native polypeptide functionality on an analytical scale (e.g., for online mass spectrometry and electrochemical sensors). − With a goal of accelerating the development of targeted and selective functionalization on a preparative scale, the remainder of this Perspective will focus on the few existing methods that strive to meet these criteria, together with key strategies that provide promise for the future development of direct electrochemical functionalization methods.…”

“…This Perspective seeks to identify and highlight the emerging potential of electrochemistry as a modality to further accelerate reaction discovery. Electrochemistry is gaining considerable momentum for small-molecule funtionalization. − However, it remains underexplored as a tool for peptide and protein modification, having, until recently, been primarily employed in an analytical capacity. − This Perspective will outline contemporary advances in electrochemical methods (primarily reported in the past three years) that have been applied to the modification of peptides and proteins on a preparative scale. The review will be organized into three sections based on the manner in which electrochemistry is used to install a given modification: (1) indirect electrochemical approaches to peptide and protein modification, (2) the use of peptide electroauxiliaries, and (3) direct, residue-specific electrochemical modification of peptides and proteins.…”

Recent developments in the cleavage, functionalization, and conjugation of proteins and peptides at tyrosine residues

“…30 Furthermore, chemical cleavage of the peptide bond usually results in the formation of modied Cor N-terminal peptide fragments, some of which are highly reactive and can be further derivatized to enable selective functionalization at peptide termini. 27,31 Despite different methods having been disclosed for Tyr-selective cleavage of peptides/proteins over a 60 year timespan, [32][33][34] there is no systematic review that covers this area. 35,36 Hence, to ll this gap, a section of this review will address the latest updates on the selective peptide/protein cleavage at Tyr sites.…”