Immobilized trypsin – enhanced MALDI-Imaging analysis of surfaces and cross-sections of works of art

Protein identification in works of art traditionally requires invasive micro-sampling, followed by protein digestion and analysis using bottom-up proteomics. While advanced MS techniques can now detect trace substances, these methods remain invasive. Recently, we developed a novel minimally invasive method for the analysis of proteinaceous materials in works of art.[1,2] This approach involves the use of a trypsin-functionalized cellulose acetate sheet, which digests proteins in situ on the surface of a work of art without any removal of material from the artifact. This method exploits the adhesion properties of fungal hydrophobins, which form a stable and homogeneous layer on the flexible sheet, allowing trypsin to be immobilized. After just 10 minutes (min) of contact at room temperature (RT), this bioactive film generates and extract sufficient peptides for unambiguous identification of the proteinaceous binder by proteomic analysis. Optical microscopy confirms that this procedure does not visibly alter the artwork or leave trypsin damaging residues.[1] A major advancement of this technique is its potential for spatially resolved imaging of surfaces or cross-sections of works of art. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has emerged in very recent years as a powerful method to extract spatially resolved chemical information in complex materials. This study proposes the first use of MALDI-MSI on a trypsin-functionalized sheet, reflecting the protein content distribution of the work of art with which it has been in contact. In brief, a 1 cm² trypsin-functionalized sheet is placed in contact with the artwork at RT for 10 min, then adhered to a glass slide for direct MALDI-MSI analysis. The analysis is performed on an Atmospheric Pressure MALDI source (TransMIT, Germany) coupled to an Orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific, Germany). The resulting MSI datasets is then processed using a home-built peptides database implemented on the Metaspace annotation platform. A series of experiments on artificially aged paint replicas will assess the method's feasibility, exploring factors such as MALDI matrix choice and deposition methods, trypsin amount and digestion conditions. Sensitivity limits in MSI acquisition related to surface heterogeneity and sample degradation/aging will also be addressed tuning instrumental parameters. The optimized method will be then applied to historical samples for validation. We aim to demonstrate that by “shaving” peptides from the surface or a crosssections of a work of art, accurate spatially resolved information about the protein content can be retrieved with minimal impact.