We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction.ESI-MS analysis of actin treated with different molar ratios of HNE (1 : 1 to 1 : 20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH(4) reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374.

Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction / G. Aldini, I. Dalle Donne, G. Vistoli, R. Maffei Facino, M. Carini. - In: JOURNAL OF MASS SPECTROMETRY. - ISSN 1076-5174. - 40:7(2005), pp. 946-954.

Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction

G. Aldini
Primo
;
I. Dalle Donne
Secondo
;
G. Vistoli;R. Maffei Facino
Penultimo
;
M. Carini
Ultimo
2005

Abstract

We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction.ESI-MS analysis of actin treated with different molar ratios of HNE (1 : 1 to 1 : 20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH(4) reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374.
4-hydroxy-trans-2-nonenal; Actin; Computational analysis; Cys374 as target residue; LC-ESI-MS/MS analysis; Michael adduction
Settore CHIM/08 - Chimica Farmaceutica
Settore BIO/06 - Anatomia Comparata e Citologia
2005
Article (author)
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/12416
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