A minimally-invasive and portable method for the identification of

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A minimally-invasive and portable method for the identification of proteins in ancient paintings Paola Cicatiello, Georgia Ntasi, Manuela Rossi, Gennaro Marino, Paola Giardina, and Leila Birolo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01718 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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Analytical Chemistry

A minimally-invasive and portable method for the identification of proteins in ancient paintings Paola Cicatiello1#, Georgia Ntasi1#, Manuela Rossi2, Gennaro Marino3, Paola Giardina1, Leila Birolo1,4* 1

Department of Chemical Sciences, University of Naples Federico II, Naples, Italy Department of Earth, Environment and Resources Sciences, University of Naples Federico II, Naples, Italy 3 University Suor Orsola Benincasa, Naples, Italy 4 Task Force "Metodologie Analitiche per la Salvaguardia dei Beni Culturali", University of Naples Federico II, Naples, Italy 2

#

these authors equally contributed to the paper

*Correspondence to: Leila Birolo, Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte S. Angelo, Via Cinthia, 80126 Naples, Italy. Phone: +39-081-679939. E-mail: [email protected].

Abstract A novel method for the analysis of proteinaceous materials present on painted surfaces was developed by taking advantage of the adhesive ability of some fungal proteins which can form a stable and homogeneous layer on flexible transparency sheets able to capture trypsin in a fully active form. We demonstrated that the bioactive sheets were able to efficiently digest proteins, present as such, on surfaces of painted tests and historical samples, releasing peptides that can allow an easy and confident identification of the proteinaceous binders by standard bottom-up proteomic approach. By this method there is no need: i) to transport the artefacts; ii) to remove, even at micro level, a sample from the object. The ingenuity of the method lies in the easily accommodated sampling coupled with a minimal invasiveness. Introduction

Proteins from ancient objects can provide key information and contribute to study the context of objects and artists and can provide invaluable information for designing restoration interventions1. Protein identification on artworks or archaeological samples is nowdays achieved by removing minute amount of the object, and then performing protein digestion followed by classical bottomup proteomics methods. Mass spectrometric data are then matched to virtual ones in the protein databases using bioinformatic softwares2. In this paper we describe a novel portable method designed to a minimally-invasive analysis of proteinaceous materials from artworks. Our approach uses a trypsin functionalized sheet of cellulose acetate to directly digest in-situ proteins on painted surfaces without the need of

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transporting the art object and avoiding any minimal removal of material from the work of art, followed then by a proteomic analysis of the released peptide mixture. The invasiveness of analytical measurements represents a central issue in conservation and restoration of cultural heritage goods and is of great concern to cultural and historical agencies. However, the level of details that proteomic approaches can provide, ranging from the type of material to the organism origin of the material itself, makes the expense of minute amount of sample fully justified in several cases. Softer protein extraction methods have been developed, starting from the development and improvement of digestion protocols in heterogeneous phase 3– 6 . These methods can be considered minimally invasive since the protease, in aqueous solution, at neutral pH, can be deposited on the surface of the sample and then gently removed without apparently and substantially affecting the sample itself. An elegant protein extraction protocol from the parchment surface was proposed that uses an electrostatic charge generated by gentle rubbing of a PVC eraser on the membrane surface7. More recently, an ethyl-vinyl acetate film functionalized with strong cation/anion exchange and C8 resins was used for the extraction of proteins and small molecules from the surface of several types of supports8–10. The method herein described takes advantage of the adhesion ability of some fungal proteins, hydrophobins11, to form a stable and homogeneous layer on a flexible transparency sheet, like those usually employed on overhead projectors, on which trypsin was immobilized. We have previously shown that the class I hydrophobin Vmh2, from the basidiomycete fungus Pleurotus ostreatus, is able to spontaneously form bioactive layers 12 on several surfaces, such as polystyrene13,14, silicon12,15 and graphene16, changing their wettability and forming extremely stable films. We have also shown that the hydrophobin layers are amphiphilic 17 and can efficiently bind functioning proteins allowing us to develop several biotools 13,14,18,19. In particular a Vmh2 self-assembled layer was used as a coating of a MALDI steel sample-loading plate, either as a simple and effective desalting method prior to MALDI analysis 20 and as a lab-onplate by immobilizing several enzymes commonly used in proteomic studies21. By taking advantage of the above experience we have included cellulose acetate sheets as a novel support surface for Vmh2 hydrophobin. We have then immobilized trypsin on the hydrophobin layer and demonstrate that the bioactive film can be used to obtain in situ, directly on the work of arts, sufficient peptides to unambiguously identify the proteinaceous binder by proteomic analyses.

Materials and Methods Pictorial and historical samples. Paint reconstructions were prepared using egg, milk, and animal glue as binders as reported in detail by Duce et al.22, and kindly provided by Prof. M. P. Colombini, University of Pisa (Italy), as also a sample from detached frescoes by Buonamico Buffalmacco (XIII century) from the Monumental Cemetery in Pisa23. Seven historical samples are unknown painting linings from the XIX-XX centuries, kindly provided by Dr. P. Somma from Laboratorio di Restauro Tele, University Suor Orsola Benincasa, Naples (Italy). Hydrophobin production ACS Paragon Plus Environment

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The protein Vmh2 was extracted from the mycelium of Pleurotus ostreatus as reported by Gravagnuolo et al., 201624. Immobilization of trypsin on Vmh2 coated cellulose acetate surface and in-situ hydrolysis A sheet of cellulose acetate was cut under hood obtaining small pieces of 2x2 cm. Then, 0.05 mL of 0.01mg/mL of Vmh2 in 60% ethanol solution was spotted on the surfaces and incubated at 60°C until the complete evaporation of solution. The pieces were washed with 0.3 mL of 60% ethanol solution to remove the excess of protein and dried at 60°C. This procedure was repeated twice before to proceed with the enzyme immobilization. 0.05 mL of 0.1mg/mL of trypsin solution in 50 mM of ammonium bicarbonate at pH 7.0, (AMBIC), were added on the coated surfaces for 5 minutes at room temperature. After the incubation, the surfaces were washed with 0.3 mL of 50 mM of AMBIC to remove the excess of trypsin and they were left to dry at room temperature. Before the use, a solution of AMBIC was sprayed on the functionalized surfaces, to moisten the surface and restore the optimal working environment of the trypsin. Hydrolysis of bovine serum albumin (BSA) on trypsin functionalized surface: 0.05 mL of 0.1 mg/mL of BSA were spotted on the functionalized surface and the reaction was carried out for 5 minutes at room temperature. Then the supernatant was removed, and the surface was dried. 0.025 mL of AMBIC 50 mM were used to extract the peptides. Hydrolysis of pictorial tests and historical samples on trypsin functionalized surface: three different pictorial tests, containing either casein or egg, or animal glue were used. They were cut into small pieces of 0.5x0.5 cm and were put in direct contact with the trypsin functionalized surface for 10 minutes at room temperature. Subsequently, the surfaces were left to dry after the removal of the pieces from the samples, and eventually, 0.025mL of AMBIC were used to extract the peptides. The peptides solution was dried under vacuum and dissolved in 0.1% of formic acid to be spotted on MALDI plate and analyzed by MALDI-TOF. The same procedure was performed to analyze the historical samples. Water Contact Angle Water contact angle (WCA) measurements were performed on a KSV Instruments LTD CAM 200 Optical Contact Angle Meter coupled with drop shape analysis software. Each contact angle was calculated as the average of two drops of 5 μL, spotted on different points of the surface. Proteomic analysis Positive Reflectron MALDI spectra were recorded on a 5800 MALDI ToF/ToF instrument (Sciex, Framingham, MA). The analyte solutions were mixed with α-Cyano-4-hydroxycinnamic acid (10 mg/mL in 70% acetonitrile and 30% of 50 mM citric acid) as the matrix. Typically, 1 μL of matrix was applied to the metallic sample plate and 1 μL of analyte was added. The mixture thus obtained was then dried at room temperature. Mass calibration was performed using external peptide standards purchased from Sciex. MS spectra were acquired from 400 to 5000 m/z, for 10000 laser shots total from randomly chosen spots per sample position. Laser intensity remained fixed for all the analyses. MS/MS analyses were performed using 1 kV collision energy with air as CID gas., and MS/MS spectra analysis was performed manually. Raw data were analyzed using the computer software provided by the manufacturers and are reported as monoisotopic masses.



Peptide mass fingerprint was carried out with a licensed version of MASCOT software (www. matrixscience.com) version 2.4.0. Proteins were identified using SwissProt database with Chordata as taxonomy restriction. MASCOT search parameters were: peptide mass tolerance 100ppm, allowed trypsin missed cleavages up to 3. No fixed chemical modification was inserted, but possible oxidation of methionine residues, and hydroxylation of prolines were considered as variable modifications. Trypsin Activity Assay Trypsin activity was measured by the method of Hummel25 using p-toluene-sulfonyl-L-arginine methyl ester (TAME) as a substrate. One unit of enzyme activity was defined as the amount of the enzyme hydrolyzing one micromole of TAME in a minute at 25°C, pH 8.1. Microscopy analysis Morphological and textural analyses of samples were carried out by means of Zeiss Axio Zoom V16 motorized microscope for large fields. The objective aperture of Axio Zoom V16 is big compared to stereomicroscopes and leads to resolution rates that are clearly better and are equipped with a system of direct drive of the zoom, with 99 % reproducibility and allows for maximum precision and accuracy for always correctly scaled images. The microscope configuration for this study is with objective Apo Z 1.5X/0.37 FWD 30mm. In particular, the images of details were acquired with: magnification of 168x, field of view of 1.4mm, resolution of 0.4 μm and depth of field of 4 μm. The acquisition of images was carried out with AxioCam ICC5 (D) microscopes camera and with Zeiss Axiovision 4 software with Z Stack and Extended Focus modules. The developed procedure include the addition of AMBIC solution through a spray (1 spray application ̴100 μL) on the biofunctionalized acetate sheet before of the contact with sample. During this time, a small amount of buffer solution (less of 100 μL) was transferred to the sample that has become damp. The microscopic analysis was performed on all analyzed samples after drying.


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Results and Discussion Recently, the class I hydrophobin Vmh2 from the basidiomycetes fungus Pleurotus ostreatus was extensively exploited to successfully functionalize several materials to be used as tools for different biotechnological applications. In this study we tried to use flexible cellulose acetate sheets, such as those used for overhead projection, as hydrophobins support surface. The actual formation of the Vmh2 layer on this cellulose acetate surface was verified by measuring the change of WCA. Upon Vmh2 deposition, drying and washing the surface, its wettability changed from hydrophobic to hydrophilic (Fig.S1), thus indicating the presence of a stable layer. Then, the modified surface was used to immobilize trypsin following a procedure already described21. After several washings with 50mM AMBIC pH7.0 to remove the excess of unbound enzyme, the support was dried and ready to use as reported in materials and methods. The workflow in Fig.1 describes, in a schematic format, the essential steps of the experimental protocol.

Figure 1: Graphical overview showing the essential steps in the experimental workflow.

Such a protocol can be considered a very user friendly one: it is ready to use, since functionalized sheets can be provided as dried to restorers and moistened just before use. Sampling can also be carried out by restores: after use, the sheet can be stored, and peptides recovered afterward just before the mass spectrometric analysis. Development and validation of the diagnostic tool. The functionalized film was in a first instance tested with a solution of bovine serum albumin (BSA), to verify the actual and active immobilization of trypsin. An aqueous solution of 0.1 mg/mL of BSA, pH 7.0, was directly, without any denaturing pretreatment, spotted on the functionalized surface, and incubated at room temperature for 10 min. The supernatant was recovered, and the peptides mixture analyzed by MALDI-TOF and compared to a control experiment where trypsin was directly added to an aliquot of the same BSA solution. The total number of identified peptides for the BSA was of 16 and 18 for immobilized and in solution trypsin, respectively, corresponding to 22% of BSA sequence coverage in immobilized condition compared with the 24% of the insolution experiment (Table S1). This result clearly demonstrates that cellulose acetate film was



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actively functionalized with Vmh2 and trypsin, and that the number of released peptides allows a confident identification of the protein. Functionalized cellulose acetate layer was tested with pictorial samples, containing the most common proteinaceous binders, i.e. casein, animal glue and egg, mixed with some common pigments (such as azurite, copper-based pigment, and St. John’s white, containing CaCO3). Three pictorial samples, containing either casein, egg or animal glue, were put in contact for 10 min at room temperature with the trypsin functionalized flexible sheet that has been made wet immediately before use. MALDI-TOF analysis of the recovered supernatants was then recorded. It is worth noting that no sample pre-treatment was carried out to simulate as much as possible an in situ portable usage. Table 1 reports the results obtained on three representative pictorial samples. To assess the quality of the results, the performances of the trypsin functionalized film were visually compared to those obtained with the conventional procedure in heterogeneous phase3, that requires microinvasive sampling of the object. The MALDI-TOF spectra are reported in comparison (Figure 2, and Table S2), and some of the signals identifying the proteins are showed in the figures. Some selected peptides were subjected to MS/MS analysis as a further confirmation, and fragmentation spectra obtained on collision-induced dissociation were manually interpreted (Figure S2-S5 as examples). In all the cases, it was possible to confidently identify the binder. The number of matched peptides makes protein identification reliable in all the cases (Table I) and guarantees a good sequence coverage. Peptide mass fingerprint in the Mascot suite, carried out with the most intense signals in the spectra, also identifies the principal components in the binders, in agreement with the simple comparison approach (Table S3).

Trypsin in Solution Immobilized Trypsin Samples

Casein + White St. John

Animal glue + azurite Egg+ azurite

Proteins (Accession number) Alpha-S1 casein (P02662) Alpha-S2 casein (P02663 Beta-casein (P02666) Collagen alpha-1(I) (P02453) Collagen alpha-2(I) (P02465) Ovalbumin (P01012)

Peptide s number 10

Sequence coverage %

Peptides number

Sequence coverage %

53

19

68

8

36

23

41

6

22

6

26

34

30

31

35

10

10

14

11

10

27

7

20

Table 1: Identification of proteins in pictorial samples by MALDI-TOF Peptide mass fingerprint. Samples were subjected to in situ digestion with the trypsin functionalized film and results were compared to those obtained with the standard protocol in heterogeneous phase3. Identification details are reported in Table S2.


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Figure 2: Comparison of the peptide mass fingerprint of pictorial samples with the trypsin functionalized film (top spectra) and the standard protocol3 in heterogeneous phase with trypsin in solution (lower spectra). Casein-based binder (A), Animal glue-based binder (B) and egg-based binder (C). ACS Paragon Plus Environment


Labels refer to identified peptides and sequences are reported for peptides that have been fragmented as further confirmation. Details of the identifications are reported in Table 1 and Table S2. Analysis of historical samples. Binder identification in ancient samples is hindered by aging, since proteins in paintings are prone to deteriorate and degrade. Several reactions occur, still not fully characterized, that can physically hinder the accessibility of proteases, such as aggregation and crosslink formation, while reactions on aminoacidic side chains introduce mass shifts that might complicate proteomic identification1,2. We tested the trypsin functionalized cellulose acetate sheets on eight different historical samples, one form the detached frescoes from the Monumental Cemetery in Pisa23 , and seven unknown painting linings from the XIX-XX centuries from the Laboratorio di Restauro Tele of the Università Suor Orsola Benincasa, Napoli. The MALDI-TOF spectra were compared to those of the painting tests reported above. Some selected peptides were subjected to MS/MS analysis and the product ion spectra obtained on collision-induced dissociation were manually interpreted as a further confirmation. The identified proteins with the detected peptides are listed in table 2 Bovine collagen alpha-1(I) and collagen alpha-2(I) was reliably identified in all the samples. Moreover, peptides from alpha S1, alpha S2 and beta caseins were detected in the sample from the Monumental Cemetery in Pisa , in agreement to previously reported results23. Peptide mass fingerprinting in the Mascot suite also confirmed our manual comparison approach (Table S3). As an example, data for the sample named “Vecchio rifodero” are reported, in comparison with animal glue-based pictorial sample. The labelled signals match to peptides from type I bovine collagen. Manual interpretation of signals at m/z 836.46 and 1427.76 (Figure S6 and S7), confirmed their attribution to peptides 1084-GPAGPQGPR-1092 from bovine collagen 1(I) (P02453) and 572-GIPGEFGLPGPAGAR-586+2Hydroxylation(P) from bovine collagen 2(I) (P02465) respectively.

Figure 3: Comparison of the peptide mass fingerprint with the trypsin functionalized film of the historical sample named as “Vecchio rifodero”, with the pictorial sample containing animal glue and azurite (lower spectra). Labels refer to identified ACS Paragon Plus Environment

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peptides and sequences are reported for peptides that have been fragmented as further confirmation (Figures S6 and S7). Details of the identification are reported in Table S4. Invasiveness of the developed methodology Invasiveness of the diagnostic method is a very relevant issue. The ability to extract proteins/peptides and/or other molecules from works of art affecting as least as possible their surface is a delicate question in the development of diagnostic tool. As a preliminary test for evaluating the impact of the analysis on the painting surface, samples were analyzed by optical microscopy to investigate if any alteration occurs upon treatment on the painting surface at the microscopic level. Figures in the supplementary (Figures S8 and S9) show the enlarged images of the surfaces of pictorial samples and historical samples before and after the analysis with the trypsin functionalized sheets. The acquired images show that the surface of the samples is not significantly altered at the microscopic level by trypsin treatment. Indeed, the loss of the small white grains that are visible in the sample named Vecchio rifodero (Fig. S9-C) occurs also after the simple water moistening (Fig. S10), and thus not because of trypsin, but because of painting support fibers become wet upon contact with the functionalized film. Moreover, in order to verify that no trypsin was released from the film onto the work of art surface, after the removal of the trypsin functionalized sheet, the surface of the pictorial sample was further washed. Within the limit of the assay sensitivity, no residual trypsin activity was observed to be present in the wash material. This result is in agreement with the observation that no trypsin activity was neither observed in a similar washing of the functionalized film, thus demonstrating that, once immobilized, trypsin is not spontaneously released in aqueous solution, and ensuring that trypsin and the painting surface interact only when they are physically in contact and no trypsin is left behind when the physical contact is discontinued.



Conclusion We demonstrated the possibility of using flexible biofunctionalized cellulose acetate sheets with trypsin to digest proteins and capture peptides from works of art with good efficiency, with a userfriendly procedure that does not visibly affect at microscopic level the surface of the material under investigation. This flexible tool is fully portable and amenable to be used in the diagnosis of paintings, valuable material and ancient archeological artifacts such as those stored in museums and private collections, without the need of micro sampling from the object. A major advantage of the developed method is that, once prepared the bioactive film, it could be used without the need to transport the works of art, and there is no need of specific technical skills to collect the sample since functionalized sheets can be provided as dried, moistened just before use and simply put in contact with the work of art. Sampling can also be carried out by restores: after use, the sheet can be stored, and peptides recovered afterward just before the mass spectrometric analysis.. The resulting samples can then be analyzed when required, since released peptides are captured onto the film surface, by a simple MALDI-TOF analysis or, eventually, by more resolutive and informative LC-MSMS analyses. The quality of the identifications is comparable to that obtained with the standard protocol that uses trypsin in solution on samples taken from the work of art. Additionally, this user friendly method is also amenable of further improvement, since the hydrophobin-coated cellulose acetate sheets can be implemented as a lab-on-plate with the noncovalent immobilization of other enzymes commonly used in protein as well as in small molecules identification/characterization, such as other proteases, glycosidases, or even lipases. For example, we are aware that a combination of N-glycosidase F and trypsin improves the identification of egg containing binders6, and we foresee that a possible introduction of Nglycosidase F together with the trypsin might improve identification of egg containing samples.

Acknowledgments

Authors want to thank Dr. Patrizia Somma from Laboratorio di Restauro Tele, Università Suor Orsola Benincasa, Naples, for providing the historical samples for the tests, and Prof. M.Perla Colombini, Dipartimento di Chimica e Chimica Industriale, Università degli Studi di Pisa, for the sample Cimitero Munumentale and the paint replicas . This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie SklodowskaCurie grant agreement No. 722606, TEMPERA (Teaching Emerging Methods in Palaeoproteomics for the European Research Area), and by grants from the University Federico II “Progetto di Ateneo IENA (Immobilization of ENzymes on hydrophobin-functionalized NAnomaterials), and POR, Parco Archeologico Urbano di Napoli (PAUN).

Declaration of interest The authors declare that they have no conflict of interest.

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A minimally-invasive and portable method for the identification of

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