Oxidation of bovine serum albumin: identification of oxidation products and structural modifications

Abstract

Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long-lived proteins. The metal-catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system. Mass spectrometry, combined with off-line nano-liquid chromatography, was used to identify modifications in amino acid residues. We have found 106 different residues oxidatively damaged, being the main oxidized residues lysines, cysteines, arginines, prolines, histidines and tyrosines. Besides protein hydroxyl derivatives and oxygen additions, we detected other modifications such as deamidations, carbamylations and specific amino acid oxidative modifications. The oxidative damage preferentially affects particular subdomains of the protein at different time-points. Results suggest the oxidative damage occurs first in exposed regions near cysteine disulfide bridges with residues like methionine, tryptophan, lysine, arginine, tyrosine and proline appearing as oxidatively modified. The damage extended afterwards with further oxidation of cysteine residues involved in disulfide bridges and other residues like histidine, phenylalanine and aspartic acid. The time-course evaluation also shows the number of oxidized residues does not increase linearly, suggesting that oxidative unfolding of albumin occurs through a step-ladder mechanism.

Identification of protein carbonylation sites by two-dimensional liquid chromatography in combination with MALDI- and ESI-MS

“…Interestingly, the identified oxidation sites of lysine and arginine did not prevent a tryptic cleavage at this position (Fig S4), although the cleavage might be slower. Similar observations have been previously reported [35][36][37].…”

“…Earlier studies on in vitro oxidized HSA and BSA identified five [44], thirty two [37] and twenty six [45] carbonylation sites, of which only five modification sites were identified in at least two different reports. In this study 14 out of the 32 carbonylation sites identified overlapped with all of the three studies mentioned above (Fig S5).…”

Food Proteomics: Mapping Modifications

“…This correlates well with observations in whole proteins under oxidative conditions Asquith and Rivett, 1969;Boreen et al, 2008;Davies and Truscott, 2001;Schäfer et al, 1997). Tryptophan and tyrosine oxidation results in a wide range of modified products, some of which can be used as oxidative markers Davies et al, 1999;Dean et al, 1997;Guedes et al, 2009;Maskos et al, 1992;Simat and Steinhart, 1998;Żegota et al, 2005). Many of these products absorb light in the visible range, such as the tryptophanderived yellow chromophore hydroxykynurenine, therefore affecting the colour of the protein material.…”

Post-translational Modifications and Mass Spectrometry Detection

“…Since the seminal work by Finch et al (209) on the identification of oxidative modifications in serum albumin, in vitro oxidative stress systems have been successfully employed to determine the mechanisms of protein oxidation (210)(211)(212)(213)(214)(215). These contributions are considered fundamental both for identifying oxidative modifications that can be considered signatures of ROS activity in biological samples and for the development of the analytical methods necessary for characterizing protein oxidation in vivo.…”