Identification of protein binders in artworks by MALDI-TOF/TOF tandem mass spectrometry T. Tripković a , C. Charvy b , S. Alves b , A.Đ. Lolić c , R.M. Baošić c,n , S.D. Nikolić-Mandić c , J.C. Tabet b a Institute for the Protection of Cultural Monuments of Serbia, Radoslava Grujića 11, 11000 Belgrade, Serbia b Equipe de Spectrometrie de masse, Institut Parisien de Chimie Moleculaire, UMR 7201, Universite Pierre et Marie Curie-Paris 6, 4 place Jussieu, 75252 Paris Cedex 05, France c Faculty of Chemistry, University of Belgrade, Studentski trg 12,11000 Belgrade, Serbia article info Article history: Received 21 December 2012 Received in revised form 21 March 2013 Accepted 28 March 2013 Available online 8 April 2013 Keywords: MALDI TOF MS LIFT-TOF/TOF tandem mass spectrometry Peptide mass fingerprint Proteinaceous binders Historical paintings abstract Aim of this work is to propose an analytical protocol for proteinaceous binder identification in paintings using tryptic peptide analysis and MALDI-TOF mass spectrometry strengthened with MALDI-TOF/TOF tandem mass spectrometry (LIFT method). Proteinaceous binders are enzymatically digested with trypsin. From each individual protein frequently occurring in binders, a specific set of peptides is releasing during enzymatic digestion giving a peptide mass fingerprint (PMF) of that particular protein. The most intensive peptide peaks in PMF were determined and annotated with their corresponding amino acid sequence by MALDI-TOF/TOF analysis and subsequent database search. Before analyzing historical painting samples, procedure was tested and optimized on several painting model samples for a reliable and efficient identification of proteinaceous materials. The method is avoiding sample manipulation as much as possible in order to reduce sample loss. Since the applied procedures led to protein identification of binding media in model samples, MALDI-TOF/TOF was for the first time applied for analysis of proteinaceous binders in old painting samples. & 2013 Elsevier B.V. All rights reserved. 1. Introduction From the ancient times artists have used various proteinaceous binders in artworks for paints, ground layers, plasters and mortars. The choice of the binding medium was dependent of painting technique and availability in artist's surrounding. Through history, different recipes, including milk, lime, and pigments were tried as well as olive oil, linseed oil, eggs, animal glue or waxes, producing varying results in terms of durability. Protein binders were the most common type of painting medium before the expansion of siccative oils. Proteinaceous substances were used not only as binders for pigments in tempera, but also as adhesives, additives for plasters and mortars and for gilding. Oil replaced tempera as the principal medium in artworks during the 15th century but proteinaceous binders remained the main medium used for panel painting and illuminated manuscripts in the South East Europe (Byzantine world) and continues to be used in Greece and Russia for Orthodox icons [1]. Knowledge about original materials present in an art object is essential for cleaning, treatment, restoration and storage as well as attribution of work of art. Various spectroscopic techniques can be used for characterization of binding media [2]. Characterization by mass spectrometry is well-suited approach because of its sensi- tivity and ability for coupling with separation techniques, as liquid or gas chromatography. Gas chromatography-mass spectrometry (GC-MS) was one of the first mass spectrometry based techniques applied for identification of protein binders in works of art and historical monuments. Amino acids were determined by GC-MS after complete hydrolysis of the proteins and their ratio or content used for protein identification in wall [3–8], easel and panel paintings [9–11] and sculptures [12–14]. Methods based on HPLC-FD (fluorescence detection) [14,15] and HPLC-DAD (UV diode array detection) [16] were also applied to analyze amino acid composition of proteins present in samples of this type. Pyrolysis GC-MS was used for identification of characteristic pyrolytic markers for different binders [17,18] and applied on determination of animal glue and casein in wall paintings [19]. Curie point pyrolysis with GC-MS has also been employed for characterization of proteinaceous media used in the artworks [20]. In recent years, proteomic approach proved to be successful in the identification of the proteinaceous components in art objects. Analysis of tryptic peptides, by bottom-up approach, is a powerful technique in conservation science for the identification of protein binders and characterization of their aging and deterioration products. HPLC-DAD is applied for analysis of tryptic peptides obtained by enzymatic hydrolysis of painting samples [21] but recently analysis were achieved using LC-MS/MS experiments [22–25]. This later approach allows undoubted identification of Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/talanta Talanta 0039-9140/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.talanta.2013.03.071 n Corresponding author. Tel.: +38 164 1673487; fax: +38 111 2639357. E-mail address: rbaosic@chem.bg.ac.rs (R.M. Baošić). Talanta 113 (2013) 49–61