PERFORM Publications

Title:	*Scavenging with TEMPO to identify peptide- and protein-based radicals by mass spectrometry: advantages of spin scavenging over spin trapping.**
Authors:	Wright PJ , English AM
Link:	https://www.ncbi.nlm.nih.gov/pubmed/12848573?dopt=Abstract
DOI:	10.1021/ja0291888
Category:	J Am Chem Soc
PMID:	12848573
Dept Affiliation:	CHEMBIOCHEM 1 Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8.

Description:

Scavenging with TEMPO* to identify peptide- and protein-based radicals by mass spectrometry: advantages of spin scavenging over spin trapping.

J Am Chem Soc. 2003 Jul 16;125(28):8655-65

Authors: Wright PJ, English AM

Abstract

The detection and characterization of radicals in biomolecules are challenging due to their high reactivity and low concentration. Mass spectrometry (MS) provides a tool for the unambiguous identification of protein-based radicals by exploiting their reactivity with suitable reagents. To date, protein-radical detection by MS has been modeled after electron paramagnetic resonance experiments, in which diamagnetic spin traps, such as 3,5-dibromo-4-nitrosobenzene sulfonic acid, convert unstable radicals to more stable spin adducts. Since MS detects mass changes, and not unpaired spins, conversion of radicals to stable diamagnetic adducts is more desirable. The use of 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) in the MS identification of protein-based radicals was explored here to establish whether scavenging via radical combination would give rise to TEMPO adducts that were stable to MS analysis. The horseradish peroxidase/H(2)O(2) reaction was used to generate radicals in derivatives of tyrosine, tryptophan, and phenylalanine as models of protein-based radicals. TEMPO(*) was added as a radical scavenger, and the products were analyzed by electrospray ionization (ESI) MS. Dramatically higher mass-adduct yields were obtained using radical scavenging vs radical trapping, which greatly enhanced the sensitivity of radical detection. The efficiency of TEMPO(*) in protein radical scavenging was examined in horse heart myoglobin and cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae. On H(2)O(2) binding to their ferric hemes, two oxidizing equivalents are transferred to the proteins as an Fe(IV)=O species and a polypeptide-based radical. In addition, CCP has been shown to reduce up to 10 equiv of H(2)O(2) using endogenous donors, thereby generating as many as 20 radicals on its polypeptide. Following myoglobin and CCP incubation with a 10-fold molar excess of H(2)O(2) and TEMPO(*), matrix-assisted laser desorption ionization (MALDI) time-of-flight analysis of the tryptic peptides derived from the proteins revealed 1 and 9 TEMPO adducts of myoglobin and CCP, respectively. Given the high scavenging efficiency of TEMPO(*) and the stability of TEMPO-labeled peptides in ESI and MALDI sources, scavenging by stable nitroxide radicals coupled with MS analysis should provide sensitive and powerful technology for the characterization of protein-based radicals.

PMID: 12848573 [PubMed - indexed for MEDLINE]

Search Publications

No results