Reactive oxygen species (ROS) can oxidize virtually all cellular components. In proteins cysteine, methionine, tryptophan, and tyrosine residues are most prone to oxidation and their oxidized forms are thus considered as biomarkers of oxidative protein damages. Ultraviolet radiation and some endogenous ROS can produce tyrosine radicals reacting with other tyrosine residues yielding intra- or intermolecular cross-links in proteins. These 3,3'-dityrosines can be quantified by their characteristic fluorescence, but analytical methods to identify the modification sites in proteins are still missing. Although mass spectrometry (MS) is routinely used to map other post-translational modifications, the analysis of dityrosines is challenged by simultaneous fragmentations of both cross-linked peptide chains producing complex tandem mass spectra. Additionally, the fragmentation patterns differ from linear peptides. Here, we studied the fragmentation behavior of dityrosine cross-linked peptides obtained by incubating three peptides (AAV. YHHFISDGVR, TEVSSNHVLI. YLDK, and LVAY. YTLIGASGQR) with horseradish peroxidase in the presence of hydrogen peroxide. Homo- and hetero-dimerization via dityrosine was monitored by fluorescence spectroscopy and MS. The fragmentation characteristics of dityrosine-linked peptides were studied on an ESI-LTQ-Orbitrap-MS using collision induced dissociation, which allowed localizing the cross-linked positions and provided generic rules to identify this oxidative modification. When human serum albumin oxidized with 50-fold molar excess of HOCl in phosphate buffer saline was analyzed by nanoRPC-ESI-MS/MS, an automatic database search considering all possible (in-silico generated) tyrosine-containing peptides as dynamic modifications revealed four different types of oxidatively modified tyrosine residues including dityrosines linking ten different Tyr residues. The automatic database search was confirmed by manual interpretation of each tandem mass spectrum.
Identification of dityrosine cross-linked sites in oxidized human serum albumin / A. Annibal, G. Colombo, A. Milzani, I. Dalle Donne, M. Fedorova, R. Hoffmann. - In: JOURNAL OF CHROMATOGRAPHY. B. - ISSN 1570-0232. - 1019(2016 Apr 15), pp. 147-155.
|Titolo:||Identification of dityrosine cross-linked sites in oxidized human serum albumin|
|Parole Chiave:||collision induced dissociation (cid); cross-linked protein; dityrosine; mass spectrometry; protein oxidation|
|Settore Scientifico Disciplinare:||Settore BIO/06 - Anatomia Comparata e Citologia|
|Data di pubblicazione:||15-apr-2016|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/j.jchromb.2015.12.022|
|Appare nelle tipologie:||01 - Articolo su periodico|