Talanta 85 (2011) 1835–1841 Contents lists available at ScienceDirect Talanta j ourna l ho me page: www.elsevier.com/locate/talanta Optimization and application of microwave-assisted acid hydrolysis for rapid quantification of protein oxidation markers using LC–MS Somaieh Afiuni-Zadeh a,b , Xinghua Guo a,∗ , Gholamhassan Azimi b , Ernst Lankmayr a a Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria b Department of Chemistry, University of Arak, 38156 Arak, Iran a r t i c l e i n f o Article history: Received 16 February 2011 Received in revised form 10 June 2011 Accepted 7 July 2011 Available online 21 July 2011 Keywords: Microwave-assisted acid hydrolysis (MAAH) Protein oxidation Protein hydrolysis Semialdehyde LC–MS a b s t r a c t Simple and efficient microwave-assisted acid hydrolysis (MAAH) of proteins was used for rapid quan- tification of -aminoadipic semialdehyde (AAS) and -glutamic semialdehyde (GGS) as major protein oxidation markers. The precursor amino acid residues corresponding to AAS and GGS in oxidized proteins were derivatized by reductive amination with sodium cyanoborohydride (NaCNBH 3 ) and p-aminobenzoic acid (ABA) followed by MAAH to generate the marker derivatives AAS–ABA and GGS–ABA. The quan- tification was performed using electrospray ionization liquid chromatography–mass spectrometry (ESI LC–MS). The important parameters for hydrolysis were optimized, which include the temperature, the reaction time, the acid concentration and volume as well as the microwave power. Compared to the con- ventional acid hydrolysis of 18–24 h using 6–12 M HCl at 110 ◦ C applied commonly in the literature and also in this work, MAAH of proteins can be completed as fast as in only 2–10 min and, additionally, with a 3–5 times higher yield of the final derivatization products. Furthermore, a better agreement between the ratio of the detected derivatization products and the theoretical yields from the studied protein has also been achieved, which indicates that MAAH may serve as a more reliable method of acid hydrolysis for this purpose than that with conventional thermal heating. The MAAH method is demonstrated to be a time-saving, reproducible and efficient technique for studying AAS and GGS as protein oxidation markers using LC–MS. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Either as functional biomolecules in biological environment or as nutrients during storage and processing of foods, proteins are vulnerable to oxidation by reactive oxygen and oxidative species. Protein oxidation can affect greatly its functionalities because exact conformation and pattern of folding are closely associated with its activity and function in a biological system [1] and food quality [2,3]. Of special interest is that recent studies have indicated that protein oxidation plays a major role in a number of human dis- eases and aging [1,4]. Oxidative damage to proteins by reactive oxygen species can result in mainly introducing carbonyl groups into amino acid residues followed by cleavage of the polypeptide backbone, cross-linking and other modification of the side chains of amino acids [5]. Reactive oxygen and nitrogen species are formed during normal metabolism and in higher fluxes under pathologi- cal conditions. Cells can detoxify some of the reactive species, e.g. by reducing protein hydroperoxides to un-reactive hydroxides [6]. Oxidized proteins are often functionally inactive and their unfold- ∗ Corresponding author. Tel.: +43 316 87332511; fax: +43 316 87332502. E-mail address: x.guo@tugraz.at (X. Guo). ing is associated with enhanced susceptibility to proteinases. Thus cells can generally remove oxidized proteins by proteolysis. How- ever, certain oxidized proteins are poorly handled by cells, and together with possible alterations in the rate of production of oxi- dized proteins, this may contribute to the observed accumulation and damaging actions of oxidized proteins during aging [1,4,7] and other neurodegenerative diseases such as Parkinson’s disease [8], Alzheimer’s disease [8,9], rheumatoid arthritis [10], amyotrophic lateral sclerosis [11] and diabetes mellitus [12]. Currently, pro- tein oxidation is increasingly gaining more attention of medical, biological and food scientists. Previously, the quantification of the total protein carbonyls through the dinitrophenylhydrazine (DNPH) method [13] with photometric detection has been the most common technique for assessing protein oxidation. However, the associated problem is that this approach does not provide any specific information about the chemical structures and formation mechanisms, which are essential evidence to understand the oxidation pathway in vivo. Two carbonyls, -aminoadipic semialdehyde (AAS) and -glutamic semialdehyde (GGS), have been recently highlighted as biomarkers of oxidative damage to proteins [14]. AAS and GGS are oxida- tive deamination products of lysine and arginine/proline residues respectively (Fig. 1). For instance, both compounds have been 0039-9140/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2011.07.050