Francesca Galluzzi

Study of protein crosslinkings in artworks using bottom up and top down proteomics

Francesca Galluzzi §; Julie Arslanoglu £; Christian Rolando §; Caroline Tokarski §

§ Miniaturization for Synthesis, Analysis and Proteomics, USR CNRS 3290, University of Lille, Faculty of Sciences and Technologies, 59655 Villeneuve d’Ascq Cedex, France; £ Department of Scientific Research, The Metropolitan Museum of Art, New York, NY, USA.

Over time, the protein materials implemented as binding media in art paintings are subjected to degradation due to ageing, environmental conditions or factors related to the painting itself. The study of chemical modifications and reticulations processes involved in the painting formulation, its evolution and its decay mechanisms remains challenging.

We propose here to study protein crosslinkings in artworks using a combination of techniques for their separation (gel-electrophoresis, size-exclusion-chromatography) and their accurate structural analysis (nanoLC-nanoESI-Orbitrap, nanoESI-FT-ICR). The methodological development was performed using 10 years-old naturally aged paint mock-up formulated with lysozyme, an egg white protein, mixed with lead white pigment (2PbCO3Pb(OH)2). Additional experiments involving UV irradiation (e.g. museum conditions) or oxidizing agents such as PbO2, H2O2 are also studied.

Various intra- and/or inter-protein crosslinkings were identified such as tyrosine-tyrosine, histidine-lysine or tryptophan-tryptophan. For example, a dityrosine crosslinking was identified between two lysozyme peptides ((RHGLDNYR)2 position 14-21) using both precursor ion mass ([M+4H]4+ at m/z 515.258) and MS/MS fragment ions (e.g. [y2α+y5β]2+ and [y2α+y2β]2+). Another example is the dityrosine crosslinking between distinct peptides; e.g. GYSLGNWVCAAK (22-33) and NTDGSTDYGILQINSR (46-61). The study was expended to other binders such as caseins and collagens showing other types of crosslinkings; e.g. glutamic acid-lysine. The developed methodology was successfully applied to historic artworks from the collection of the Metropolitan Museum of Art showing various types of crosslinkings; e.g. IVSDGNGMNAWVAWRNR (lysozyme peptide 98-112) W11-14 intracrosslink identified in a early 1490s tempera-on-wood from Lorenzo di Credi. This presentation will discuss methodologies and results obtained from the various studied samples.