Collagen-based proteinaceous binder-pigment interaction study under UV ageing conditions by MALDI-TOF-MS and principal component analysis Julia Romero-Pastor, a Natalia Navas, b * Stepanka Kuckova, c,d Alejandro Rodríguez-Navarro a and Carolina Cardell a This study focuses on acquiring information on the degradation process of proteinaceous binders due to ultra violet (UV) radiation and possible interactions owing to the presence of historical mineral pigments. With this aim, three different paint model samples were prepared according to medieval recipes, using rabbit glue as proteinaceus binders. One of these model samples contained only the binder, and the other two were prepared by mixing each of the pigments (cinnabar or azurite) with the binder (glue tempera model samples). The model samples were studied by applying Principal Component Analysis (PCA) to their mass spectra obtained with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). The complementary use of Fourier Transform Infrared Spectroscopy to study conformational changes of secondary structure of the proteinaceous binder is also proposed. Ageing effects on the model samples after up to 3000 h of UV irradiation were periodically analyzed by the proposed approach. PCA on MS data proved capable of identifying significant changes in the model samples, and the results suggested different aging behavior based on the pigment present. This research represents the first attempt to use this approach (PCA on MALDI-TOF-MS data) in the field of Cultural Heritage and demonstrates the potential benefits in the study of proteinaceous artistic materials for purposes of conservation and restoration. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: MALDI-TOF-MS; principal component analysis; pigment-proteinaceous binder interaction; FTIR protein conformational study; collagen INTRODUCTION The use of organic materials in artworks is well known; for example, substances like oils, gums, proteins or waxes are tradi- tional binders in diverse painting techniques. [1,2] In particular, proteinaceous binders have been used since antiquity, e.g. egg, glue from animal skin, or milk in so-called tempera painting. [3,4] Although some research has focused on the identification of the different types of proteins, the ageing processes of proteinaceous paint binders are little studied due to their complex composition and structure. [5–11] Moreover, few studies have examined the role of inorganic pigments in the ageing processes of these binders. [11–14] Particularly, the characterization of proteinaceous binders by GC analysis of amino acids has demonstrated changes in the amino acid compositions due to the presence of pigments. [12,13] Specifically, alkyl and imino-substituted amino acids were much less affected by pigments and ageing than other amino acids. [12] The presence of calcium carbonate caused a decrease in both amino acid asp and glu in egg and milk tempera, while no effects were observed for the glue tempera. [13] Up to now, the study of proteinaceous binders has been mainly carried out by Liquid Chromatography (HPLC), Gas Chromatography, Fluorescence Spectroscopy, Raman Microscopy (RM) or Fourier Transform Infrared Spectroscopy (FTIR). [2,5,8,9,11,15] These chromatographic methods characterize the proteinaceous binders through the determination of the derivatized amino acids previously obtained by hydrolysis of the proteinaceous material. [4–7,12,13] Commonly, the use of FTIR to study proteins has been applied to gain molecular and conformational informa- tion via the observed shift of the amide I maximum. Hydrogen bonds linking the peptide bond NH of a glycine residue with a peptide carbonyl (C O) group in an adjacent polypeptide help hold the triple-helical structure of collagen. [16] These bands, specifically the amide I band, are conformationally sensible, and are often used to determine protein secondary structure caused by intramolecular and intermolecular hydrogen bonding of amide groups. [17–20] In particular, the amide I band (carbonyl stretching vibrations) * Correspondence to: Natalia Navas, Department of Analytical Chemistry, University of Granada, Campus Fuentenueva s/n, 18071 Granada, Spain. E-mail: natalia@ugr.es a Department of Mineralogy and Petrology, University of Granada, Campus Fuentenueva s/n, 18071 Granada, Spain b Department of Analytical Chemistry, University of Granada, Campus Fuentenueva s/n, 18071 Granada, Spain c Charles University, Department of Chemistry and Chemical Education, M.D. Rettigové 4, 116 39 Prague 1, Czech Republic d Institute of Chemical Technology Department of Biochemistry and Biotechnology, Technická 5 166 28, Praha 6, Czech Republic J. Mass. Spectrom. 2012, 47, 322–330 Copyright © 2012 John Wiley & Sons, Ltd. Research Article Received: 4 July 2011 Revised: 27 January 2012 Accepted: 30 January 2012 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.2966 322