RESEARCH PAPER Identification of lipid- and protein-based binders in paintings by direct on-plate wet chemistry and matrix-assisted laser desorption ionization mass spectrometry Cosima Damiana Calvano & Inez Dorothé van der Werf & Francesco Palmisano & Luigia Sabbatini Received: 10 October 2014 /Revised: 18 November 2014 /Accepted: 19 November 2014 /Published online: 30 November 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Direct on-target plate processing of small (ca. 100 μg) fragments of paint samples for MALDI-MS identifi- cation of lipid- and protein-based binders is described. Frag- ments were fixed on a conventional stainless steel target plate by colloidal graphite followed by in situ fast tryptic digestion and matrix addition. The new protocol was first developed on paint replicas composed of chicken egg, collagen, and cow milk mixed with inorganic pigments and then successfully applied on historical paint samples taken from a fifteenth century Italian panel painting. The present work contributes a step forward in the simplification of binder identification in very small paint samples since no conventional solvent ex- traction is required, speeding up the whole sample preparation to 10 min and reducing lipid/protein loss. Keywords Painting . Colloidal graphite . MALDI . Lipid . Protein Introduction Paint layers of polychrome works of art are typically made up of colored pigments and a medium or binder such as drying oil, egg, animal glue, casein, and vegetal gum. Identification of the original binder components through their constitutive molecules (e.g., lipids, proteins, polysaccharides, etc.) and investigation of their modifications induced by ageing pro- cesses are of primary importance for the conservation and preservation of paintings and other polychrome artefacts. For instance, the characterization of organic materials in artis- tic paintings is typically performed by gas and liquid chroma- tography (GC and LC) or pyrolysis-GC [1] that, however, require several pre-analytical steps including laborious methods of hydrolysis and derivatization. The introduction of soft ionization (I) methods like matrix- assisted laser desorption (MALD) and electrospray (ES) has greatly contributed to the explosion of mass spectrometry (MS)-based techniques in the field of the omics sciences. As an obvious consequence, MS-based proteomics studies started to be increasingly appreciated also in the field of cultural heritage where non-destructive techniques are typically pre- ferred and the sample amount for classical analysis is drasti- cally limited. Such a limitation can be easily faced by the inherently high sensitivity of MALDI or nano-LC-ESI-MS as demonstrated by several papers reporting protein identifica- tion by the classical bottom-up approach [2–4]. Sample pre- treatment is generally quite laborious: proteins are extracted, making use of appropriate alkaline or acidic media, and then submitted to processes such as denaturation/reduction/alkyl- ation and finally enzymatic (usually tryptic) digestion. Other- wise, digestion with trypsin may be directly performed on paint fragments or cross sections, followed by purification of the peptide solution before MALDI or nano-LC-ESI-MS analysis [5, 6]. Although MALDI-MS has been widely used in the field of cultural heritage for protein analysis, its application to non- proteinaceous materials is much more limited and has been reported in only a few studies. For instance, van den Berg et al. [7, 8] have investigated the oxidative ageing of drying oil, Published in the topical collection celebrating ABCs 13th Anniversary . C. D. Calvano : I. D. van der Werf (*) : F. Palmisano : L. Sabbatini Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via Orabona 4, 70125 Bari, Italy e-mail: inezdorothe.vanderwerf@uniba.it C. D. Calvano : F. Palmisano Centro Interdipartimentale S.M.A.R.T., Università degli Studi di Bari Aldo Moro, Via Orabona 4, 70125 Bari, Italy L. Sabbatini Centro Interdipartimentale “Laboratorio di ricerca per la diagnostica dei Beni Culturali”, Università degli Studi di Bari Aldo Moro, Via Orabona 4, 70125 Bari, Italy Anal Bioanal Chem (2015) 407:1015–1022 DOI 10.1007/s00216-014-8359-6