Efficient and Selective Chemical Labeling of Electrochemically Generated Peptides Based on Spirolactone Chemistry

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. Cleavage of the peptide bond following electrochemical oxidation of Tyr or Trp results in a spirolactone moiety at the newly formed C-terminus offering a handle for chemical labeling. In this work, we developed a highly efficient and selective chemical labeling approach based on spirolactone chemistry. Electrochemically generated peptide-spirolactones readily undergo an intramolecular rearrangement yielding isomeric diketopiperazines precluding further chemical labeling. A strategy was established to prevent intramolecular arrangement by acetylating the N-terminal amino group prior to electrochemical oxidation and cleavage allowing the complete and selective chemical labeling of the tripeptide LWL and the decapeptide ACTH 1-10 with amine-containing reagents. As examples, we show the successful introduction of a fluorescent label and biotin for detection or affinity enrichment. Electrochemical digestion of peptides and proteins followed by efficient chemical labeling constitutes a new, powerful tool in protein chemistry and protein analysis.

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

“…18−20 We previously developed an approach to label electrochemically cleaved peptides via spirolactone chemistry to introduce affinity tags 20 for subsequent enrichment by immobilized avidin chromatography and characterization by mass spectrometry. In this approach, acetylation of the amino group at the N-terminus was essential to prevent intramolecular rearrangement to nonreactive diketopiperazines.…”

“…EC-LWL with Cu(II) was prepared by adding 4 μL of Cu(II) chloride dehydrate (100 μM) into 2 mL of solution and dried following the same procedure as described in our previous work. 20 EC-LWL without Cu(II) was dried following the same procedure as the control.…”

“…10−13 Specific cleavage of the peptide bond C-terminal to tyrosine (Tyr) and tryptophan (Trp) residues occurs after electrochemical oxidation of peptides and proteins, which makes electrochemistry (EC) a potential instrumental alternative to chemical and enzymatic peptide bond cleavage. 14−20 However, EC oxidation yields complex mixtures due to the generation of noncleavage oxidation products in addition to cleaved peptides, which poses a problem for proteomics applications.…”

“…25−27 Electrochemical cleavage of the peptide bond of Tyr or Trp yields a spirolactone moiety at the newly formed C-terminus providing a handle for specific chemical labeling. 17,20 In previous work, we used this unique spirolactone to introduce affinity tags but encountered problems with spirolactone stability and intramolecular rearrangement. 20 …”

“…17,20 In previous work, we used this unique spirolactone to introduce affinity tags but encountered problems with spirolactone stability and intramolecular rearrangement. 20 …”