Method for Noninvasive Analysis of Proteins and Small Molecules

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A New Method for Non-Invasive Analysis of Proteins and Small Molecules from Ancient Objects Marcello Manfredi, Elettra Barberis, Fabio Gosetti, Eleonora Conte, Giorgio Gatti, Clara Mattu, Elisa Robotti, Gleb Zilberstein, Igor Koman, Svetlana Zilberstein, Emilio Marengo, and Pier Giorgio Righetti Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03722 • Publication Date (Web): 08 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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Analytical Chemistry

A New Method for Non-Invasive Analysis of Proteins and Small Molecules from Ancient Objects Marcello Manfredi1,2 $, Elettra Barberis1,2 $, Fabio Gosetti1, Eleonora Conte2, Giorgio Gatti1, Clara Mattu3, Elisa Robotti1, Gleb Zilberstein4, Igor Koman5, Svetlana Zilberstein4, Emilio Marengo1*, Pier Giorgio Righetti6* 1

Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale, viale Teresa Michel 11, 15121, Alessandria, Italy. 2 ISALIT , Via G. Bovio 6, 28100, Novara, NO, Italy. 3 Politecnico di Torino - sede di Alessandria, Viale T. Michel, 5 15121 Alessandria, Italy. 4 Spectrophon Ltd, Pekeris 4, Rehovot, 76702, Israel. 5 Translational Medicine Institute, Ariel University, Israel 6 Department of Chemistry, Materials and Chemical Engineering ‘‘Giulio Natta’’, Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy. $

These authors contributed equally to this work. Corresponding author: Prof. Emilio Marengo, email [email protected] - tel. +390131360259 * Corresponding author: Prof. Pier Giorgio Righetti, email [email protected] - tel. *

+390223993045 ABSTRACT Proteins and small molecules from ancient objects and cultural heritage can provide key information and contribute to study the context of objects and artists. However, all presentday protocols and strategies for the analysis of ancient samples are often invasive and require micro sampling. Here, we present a new method for the non-invasive analysis of proteins and small molecules: the technique uses a special ethyl-vinyl acetate film functionalized with strong cation/anion exchange and C8 resins, for interacting with both proteins and small molecules present on the surface of the objects, followed by LC-MS/MS analysis. The new method was fully validated for the determination of both proteins and small molecules on several types of supports, showing excellent analytical performances such as for example R2 of the calibration curve of 0.98 and 0.99 for proteins and small molecules, low but very repeatable recoveries, particularly adequate for investigations on precious ancient samples that must not be altered by the analytical procedure. ESEM images and LED multispectral imaging confirmed that no damages or alterations occurred onto the support surfaces and no residues were left from the extractive film. Finally the new method was applied for the characterization of the binders of a historical fresco of the XVI century from the Flemish painter Paul Brill, and of a recently discovered fresco from Isidoro Bianchi (XVII century). Moreover the method was employed for the identification of the colorant used by Pietro Gallo (XIV century) on a wood panel. The method here reported can be easily applied to any other research on ancient precious objects and cultural heritage, since it does not require micro sampling and the proteins/small molecules extraction can be performed directly in situ, leaving the object unchanged and intact. KEYWORDS: non-invasive extraction; proteins; small molecules; ancient objects; nondestructive analysis; cultural heritage; LC-MS/MS ACS Paragon Plus Environment


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INTRODUCTION Recent technological developments in proteomics and metabolomics spurred the analysis of historical, archaeological and paleontological objects. Identification of proteins and small molecules from cultural heritage objects is important for the characterization of the materials used by the artists or to investigate the context of the object. The application of analytical chemistry to archaeological research today represents a major methodological subfield within the archaeological sciences.1 Although the advent of high performance mass spectrometers allows the identification and quantification of low amounts of substances from small samples, most of the actual protocols and strategies are invasive and usually require micro sampling.2 Proteinaceous materials derived from animal products and tissues (eggs, milk, skin, bones, etc.) have been widely used in cultural heritage as binders and adhesives. Their accurate identification is crucial for studying the context of objects and fundamental to improve the conservation practices. Proteomics approaches are often employed for the identification of protein binders in cultural heritage objects, in particular in historical paints3, frescoes4,5 and polychrome pottery.6 Reliable protein extraction and identification from oldage parchment documents had already been performed by sampling tiny fragments,7 while parchment proteins were non invasively extracted by using an electrostatic charge generated by the gentle rubbing of a PVC eraser.8 The in situ analysis of proteins has been investigated using desorption electrospray ionization mass spectrometry (DESI-MS), thus obtaining significant results and good preservation of the protein material9. The identification of proteins allowed also a better understanding of human history: the use of shotgun proteomics on a 500-year-old Andean mummy provided for the first time the evidence of active pathogenic infection in an ancient sample10. Conventional analytical techniques used to identify and analyze organic substances present in artworks include Enzyme Linked Immunosorbent Assay (ELISA). The methods applied for the analysis of proteinaceous media still suffer from the problem of the large amount of sample required and the sensitivity to the contamination with other proteins 11, 12 . Infrared spectroscopy coupled to chemometrics methods, like PCA and KNN13-15, as widely used for the analysis of cultural heritage samples, was able to classify different binding media. Notwithstanding the great performance of new portable instruments, the analysis is still challenging due to the infrared spectra complexity and low repeatability. Over the centuries, artists used organic colorants on paper as inks, on wall for mural paintings, on wood panels and canvas. The characterization of these small molecules is very important in order to study the painting techniques or for dating an artwork. Liquid chromatography coupled to mass spectrometry (LC-MS) plays an important role in the analysis of historical organic colorants but all the available protocols require a microsample for the analysis16. The non-invasive approach, like visible fiber optics reflectance spectra and laser ablation surface-enhanced Raman micro-spectroscopy, can give preliminary results, but mass spectrometry is still necessary for confirmation17, 18. A recent work of Lech et al.19 clearly showed that tandem mass spectrometric detection coupled to high-performance liquid chromatography is essential for the unequivocal identification of Polish cochineal, even if 0.2–0.3 mg of fiber was used for the colorant extraction. Based on the recent scientific literature in this field all the developed analytical procedures require at least a micro sampling from the object20-33. However, non-invasive instruments and techniques are always preferred for the analysis of precious and unique objects34-37, especially in the cultural and archeological fields. All these challenges make the detection of proteins and small molecules in art very difficult. To this aim in this paper we present a new method for the non-invasive analysis of proteins and small molecules. A special ethyl-vinyl acetate (EVA) film functionalized with ACS Paragon Plus Environment

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strong cation/anion exchange and C8 resins was prepared, characterized with the environmental scanning electron microscope (ESEM) and used for the extraction of proteins and small molecules from the surface of several types of supports. The extracts were then analyzed by LC-MS/MS analysis. The method was analytically validated and the ESEM and LED multispectral images confirmed that no damage or change affected the support surfaces and no residues were left from the application of the extractive film. Finally the method was applied for the characterization of the binders of a historical XVI century fresco from the Flemish painter Paul Brill and of a recently discovered fresco from Isidoro Bianchi (XVII). Moreover, the method was employed for the identification of the colorant used by Pietro Gallo (XIV century) on a wood panel. EXPERIMENTAL SECTION Materials. Methanol (LC-MS Ultra CHROMASOLV, >99.9%), 2-propanol (LC-MS Ultra CHROMASOLV, >99.9%), carminic acid (>96%), Alizarin (>97%), Indigo (>95%), acetic acid (eluent additive for LC-MS, >99.9), ammonium bicarbonate (AMBIC), dithiothreitol (DTT), iodoacetamide (IAA), trypsin, formic acid (FA, eluent additive for LC-MS, >99.9), ammonium acetate, trifluoroethanol TFE (>99%), ammonium hydroxide, bovine serum albumin, and water (LC-MS Ultra CHROMASOLV, >99.9%) were purchased from SigmaAldrich (Milwaukee, USA). The ultrapure water was obtained through a Millipore Milli-Q system (Milford, USA). The 1000.000 mg L-1 stock standard solution of carminic acid, alizarin and indigo was prepared in ultrapure water, diluted as required in ultrapure water and preserved at 4°C in dark glass vials. The mixed-bed cation (SCX)/anion (SAX) exchange resins AG501 and C8 resins were from Bio-Rad (Hercules, USA). Synthesis and characterization of the EVA film. A special plastic-like film based on ethyl-vinyl acetate (EVA) as binder of ground AG 501 mix-bed cation/anion exchange and C8 resins (all from Bio Rad) was prepared. A mixture was made comprising 70% 1-10 µm size ground beads and 30% EVA (the melting temperature was 75°C). This mixture of melted EVA and Bio-Rad resins was poured in a "Brabender" mixer W30 and extruded via a "Brabender" extruder KE19 (both from Brabender GmbH, Duisburg, Germany) in the form of a thin film. The thickness of the film was 150-200 µm. These plastic films were wetted in doubly distilled water prior to their applications to the surface of the specimens under investigation. The contact time was 10-15 min. The morphologies and elemental mapping of the functionalized EVA film were then characterized with an environmental scanning electron microscopy (Quanta 200, FEI Company, Eindhoven, The Netherlands) equipped with EDX (EDAX Inc., Mahwah, USA). Canvas, painting, fresco, bone, parchment and linen samples. Replicas of canvases, paintings on wood, “fresco” and “secco” mural paintings were prepared. The panel of different supports, binders and colorants was designed by following the ancient receipts used by the most important artists38-40 in order to simulate only real cases. The canvas support was prepared with linen, rabbit skin glue 1:14 in water and ground gypsum, while the binder was fresh whole egg. The painting was prepared using a wood panel spread with bovine glue, linen with rabbit skin glue 1:14 in water and ground gypsum, while the binder was fresh whole egg. The fresco mural painting was produced with lime paste, large and fine granules of sand, red ochre, ultramarine blue and yellow ochre. The colors were spread in lime and protein binders on the wet plaster. The “secco” mural painting was also set up by drying the intonaco before the application of the color. For the fresco rabbit skin glue (1:14 in water) and ammonium caseinate (15% milk casein + 82% of water and 3% of NH4OH) were employed as binders. For the “secco” mural painting egg yolk and egg white were used as binders. The bovine bone was a scapula that was carefully ACS Paragon Plus Environment


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washed and heated for 10 hours at 65°C in an oven. The parchment sample was of bovine origin and the linen was pure. Protein and colorant calibration curves, extraction and digestion. The calibration curve of Bovine Serum Albumin (BSA) was carried out by spreading 100.0 µL of BSA solution at 1.00, 0.50, 0.10, 0.05 and 0.01 µg/mL on five different areas of 1x1 cm size on a painting on wood. The calibration curve of egg tempera (fresh egg incorporated with the pigment Ultramarine Blue) was obtained by spreading 100.0 µL of the solution at 1.000, 0.250, 0.125, 0.025 and 0.003 dilutions from fresh egg on five different areas of 1x1 cm size on a painting on wood. The calibration curve of carminic acid, alizarin and indigotin was constructed by spreading 100.0 µL of each solution at 100.0, 50.0, 10.0, 5.0, 2.5 and 1.0 µg/mL on different areas of 1x1 cm size on a painting on wood. The protein and colorant extractions for the calibration curves and from the historical frescoes were carried out with 1x1 cm strips while the extractions from other replicas were carried out with smaller ones (3x5 mm). Extraction protocol. The strip was gently humidified with ultrapure water and then placed in contact with the surface for 30 minutes. The proteins were eluted from the strip with 500 µL of 1.0 M ammonium acetate in an eppendorf for 30 minutes. Then, the strip was removed and the proteins were denatured with TFE at 60 °C, reduced with 200.0 mM DTT, alkylated with 200.0 mM IAM and digested with trypsin overnight. The colorant was eluted from the strip with 500.0 µL of a mixture of methanol and 0.1% of formic acid. Micro-sampling analysis. Two micro samples of about 4 mg were collected from the model fresco mural painting and from the model canvas. Then proteins were denatured in AMBIC (200 µL) with TFE at 60 °C, reduced with 200.0 mM DTT, alkylated with 200.0 mM IAM and digested with trypsin overnight. The supernatant was then recovered for the LCMS analysis. Description of historical samples. For this research we have selected two frescoes of the XVI century, whose images are shown in Fig. 2A (Flemish painter Paul Brill (15541626)) and 2B (a recently discovered fresco from Isidoro Bianchi (1581-1662)). The two frescoes are situated in the Parella castle in Italy (XIII century), which is decorated with precious allegorical frescoes recently attributed to the baroque painter Isidoro Bianchi and to the Flemish artist Paul Brill, who painted several artworks conserved today in the Vatican Museums. A wood panel of the XIV century from the artist Pietro Gallo, which is shown in Fig. 2C, was also analyzed with the EVA film. The wood panel is situated in the Palazzo Madama Museum in Turin (Itay). Pietro Gallo from Alba was an Italian medieval painter. Its activity is documented in Alba and Genoa between the second half of the XIV century and the 1401 (year of death). He was strongly influenced by the famous Barnaba from Modena (Genova). LC-MS/MS and multispectral imaging experimental details are provided in the Supporting Information. RESULTS Characterization of the EVA film. The film with embedded beads was analyzed with an environmental scanning electron microscope (ESEM) in order to characterize its surface. The distribution of the trapped resins was analyzed for assessing their chemical behavior and their ability to adsorb ACS Paragon Plus Environment

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proteins and small molecules. Figure 1 shows the ESEM images of the EVA strip: the surface roughness, the morphology of the material and the presence of 10 µm-size particles quite uniformly distributed can be appreciated (fig. 1a). The material of the strip is porous as it can be noted in the cross-section in figure 1b. Given the fact that the film contains three different types of beads, it was of interest to investigate if they were uniformly distributed on its surface or if there were clusters of some types of resins. In particular, we wanted to ascertain if there could have been aggregates of the anion and cation exchange resins, since if they were bound via salt bridges among the positively and negatively charged residues, this would hamper their ability to harvest proteins and metabolites from the specimens under analysis. Panels C to E in Figure 1 indeed show that such aggregates do not exist. Other aspects to be mentioned: crushing of the beads is a preferred procedure, since this enhances the surface area of the capturing elements and thus favours better harvesting of analytes. Development and validation of the extraction method. One of the most important issues in the analysis of historical and precious objects is the non-invasive identification of the material composition. A very efficient method of analysis should enable the extraction, the identification, and the quantitative estimation of major and trace components while leaving unchanged the original sample. Here, the suitability of the EVA film for the quantitative analysis was evaluated for both proteins and small molecules. The validation of the method was carried out on the proteins BSA and Ovalbumin from egg tempera (to simulate real cases) and on the small molecules carminic acid, alizarin and indigotin. The solutions of the investigated binders and colorants were spread on different areas of 1x1 cm size on a painting on wood and were extracted with the EVA film. The colorants were directly analyzed with LC-MS while proteins were digested into peptides prior the analysis, as detailed in the Supporting Information. For each analyte a calibration plot reporting the peak area of the “quantifier” transition signal (y) versus standard concentration (x) was built. Five concentration levels in the range between 0.005-1.000 µg mL-1 for BSA and 0.003-1.000 dilution from fresh egg for Ovalbumin and six concentration levels in the range between 1.00-100.00 µg mL-1 for carminic acid, alizarin and indigotin were considered. Moreover, to overcome possible memory effects, the standard solutions were injected in randomized order. For all the analytes a linear regression fit with a weighting factor 1/x was used and a good linearity was obtained: the regression coefficients were 0.9866 for BSA, 0.9959 for Ovalbumin, 0.9989 for carminic acid, 0.9983 for alizarin and 0.9927 for indigotin. The limit of detection (LOD) was calculated as the concentration of the analyte that gives a signal (peak area) equal to the average background (Sblank) plus three times the standard deviation sblank of the blank (LOD = Sblank + 3sblank), while the limit of quantification LOQ is given as LOQ = Sblank + 10sblank. The LOD and the LOQ for the protein were calculated as 0.001 µg mL-1 and 0.004 µg mL-1, respectively for both BSA and Ovalbumin. As concerns the dyes, LOD and LOQ were calculated as 0.30 µg mL-1 and 1.01 µg mL-1 for carminic acid, 0.28 µg mL-1 and 0.93 µg mL-1 for alizarin and 0.46 µg mL-1 and 1.54 µg mL-1 for indigotin as reported in Table 1. The LODs and LOQs of the method are very good considering that the extraction of the analytes was performed on a painting on wood and that the egg tempera sample simulates a real case. The inter-day precisions on concentration were evaluated by analyzing the analytes every day (five replicates) for six days. The results show that the inter-day precisions are 6.88% and 7.32% for BSA and Ovalbumin and 8.37%, 6.32% and 9.71% for carminic acid, alizarin and indigotin respectively.

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To evaluate the recovery R (%) of each analyte and to verify its possible dependence on the concentration, the analyte standard solutions at different concentration levels used for the calibration curve were analyzed. The recovery values were calculated as Cobs/Cref where Cobs is the difference between the concentration determined for the spiked sample and the native concentration in the same sample, and Cref is the spiked concentration. For all the analytes an average percentage of recovery R(%) was therefore calculated and reported in Table S1. As it can be observed the R (%) values for BSA ranges from 3.97% to 9.36% while the R (%) values for Ovalbumin ranges from 4.01 % to 11.31%. The recoveries of the various dyes range from 0.010% to 0.83% for carminic acid, from 0.15% to 2.23% for alizarin and from 0.03% to 2.10% for indigotin. It may be remarked that the low but reproducible recovery is particularly indicated for the heritage field because the extraction method can be considered non-invasive, especially for the small molecules, which often represent the most interesting substances in cultural heritage materials. Moreover, the ability of detecting proteins and small molecules at the sub-nanomolar and micromolar concentrations (140 picomolar for BSA and 2.03 micromolar for carminic acid) on a surface is very encouraging to explore the methodology for detecting other biological and organic substances in cultural heritage objects and surfaces. We have next investigated two fundamental aspects of this novel methodology, namely that the EVA film, once applied to a given object, would not leave resin residues on its surface or imbedded into its fibers and also, in the case of frescos or paintings, that it would not remove precious material, thus permanently damaging it. To that aim, the characterization of the strip surface before (Figure S-1a) and after (Figure S-1b) its use for the extraction of proteins from a fresco was carried out: as it can be noted, no visible residues from the fresco appear on the strip surface. Moreover, the parchment surface before (Figure S-1c) and after (Figure S-1d) was also investigated with the ESEM microscope in order to detect possible residues that may have been deposited on it by the EVA film. Figure S-1e shows the parchment sample with some chromatographic beads deposited on the surface. The ESEM images confirm that the strip did not leave any residues on the parchment support. Additionally, the LED multispectral imaging, which measures quantitatively the reflectance spectra of a surface, was performed on a parchment sample colored with carminic acid. The imaging analysis of the same area before and after the dye extraction with the EVA film showed that the reflectance spectra did not change after the application of the method (Support information Fig. S-2). All these data, together with the low recovery measured for both proteins and small molecules, confirmed the non-destructivity of the analytical method developed here and that it leaves unchanged the analyzed surface. Method comparison. The performances of the EVA film method were compared with a traditional procedure that requires a micro sampling from the object. The protein extractions from fresco mural paintings and canvases were compared: Table S2 reports the proteins identified in the samples using the two methodologies. The invasive procedure allowed the identification of the main five milk proteins (Alpha-S1-casein, Beta-casein, Kappa-casein, Alpha-S2casein, Beta-lactoglobulin) while the EVA method was able to identify only Alpha-S1casein and Beta-casein. The most important aspect is that the invasive method left a 2x2 mm hole on the surface of the fresco, while when using the EVA film the artwork was not damaged. Moreover the identification of the two milk proteins extracted with the EVA is sufficient to detect the binder. The lower performance of the EVA method is due to the fact that in the mural painting the binders are embedded in the intonaco, therefore the extraction is more difficult. However, the protein extraction from canvas allowed the identification of all the major egg (Ovalbumin, Ovomucoid, Ovotransferrin, Vitellogenin-1, ACS Paragon Plus Environment

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Vitellogenin-2, Ovoinhibitor, Ovalbumin-related protein Y, Serum albumin, Lysozyme C, Ovalbumin-related protein X and Riboflavin-binding protein) and glue (Collagen alpha-2(I) chain and Collagen alpha-1(I) chain) proteins. However, with the invasive analysis we were able also to identify other less abundant egg and glue proteins. Application of the method to several supports. The efficacy of the EVA film in extracting macromolecules as well as metabolites and small molecules from a variety of specimens is here reported. In particular Table S3 lists the proteins that could be extracted and identified via MS/MS from different samples, such as canvasses, painting on wood, “fresco” mural painting as well as “a secco” murals and finally from bovine bone and parchment. It can be appreciated that a fairly large number of proteins could be recognized with high confidence, those of high-abundance with a higher number of peptides (for instance as many as 42 peptides for ovotransferrin and 20 for ovalbumin for those binders that contain egg) and a minimum of two peptides for those species present in low-abundance (e.g., cystatin, vitelline membrane outer layer protein 1, lysosomal-trafficking regulator and interferon gamma for painting on wood). Interestingly, even from an old and dry bovine bone as many as 26 different proteins could be extracted in sufficient amounts to permit their characterizations via mass spectrometry. Even more strikingly, one protein (Collagen alpha-1(I) chain) with a good Mascot score of 644 could be extracted from a bovine parchment and authenticated with eleven peptides. This nondestructive analysis compares favorably with the data of Toniolo et al.7, where eight proteins (four types of collagen, type VI, alpha 3-like isoform 3, alpha-1(XIV) chain-like, alpha-2(I) chain precursor, alpha-1(I) chain precursor, mimecan, histone H2A type 3, protein disulfide isomerase and tubulin alpha-1C chain) could be identified in a 800 year old parchment, however at the cost of destroying and digesting a fragment of this valuable item. In the fresco mural painting the method was able to identify both the collagen proteins (Collagen alpha-1(I) chain and Collagen alpha-2(I) chain) and the casein proteins (AlphaS1-casein and Beta-casein). Moreover, as reported in Table S1, the LC-MS analysis on the “secco” mural painting samples was able to discriminate between the use of egg yolk (Vitellogenin-2) and egg white (Lysozyme C, Ovalbumin and Ovotransferrin) as binders. Table S4 shows that also small molecules can be extracted and quantified from different materials, such as parchment, linen and painting on wood. In this particular case, carminic acid dye could be assessed even at rather low levels, such as ng/µL. It should be also highlighted that the proteins and colorant extractions from all these supports were carried out with a very small-size strip of 3x5 mm, confirming the excellent sensitivity and the great performance of the method. Analysis of historical frescoes and wood panel. Another important aspect of the EVA film technology is its ability to extract proteins and/or other molecules from ancient frescoes without damaging their surface. Two frescoes of the XVI century represented in Fig. 2A and 2B were analyzed with the EVA film: in both cases different types of caseins have been identified, as reported in Table 2. In the insert of Fig. 2B, where the gloved finger is pointing, one can see, underneath it, the EVA film diskette applied to the surface of the fresco. Even if in the fresco the binder is embedded in the intonaco, and in consequence the extraction from the support is harder than in other supports like canvas, the beta-casein protein was identified with three peptides in both the samples. In the fresco from Isidoro Bianchi we also identified Alpha-S1-casein and Kappacasein with one peptide each one. The peptide GPFPIIV from beta-casein was identified in the two historical frescoes but not in the model one using the non-invasive EVA extraction. But the same peptide was ACS Paragon Plus Environment


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identified using the traditional invasive method. We have to consider that the two frescoes are five hundred years old and the proteins may have undergone degradation. Moreover, the simultaneous presence of several organic and inorganic materials, the environmental and ageing contamination and the low amount of milk used in the fresco preparation may complicate the proteins extraction. A wood panel of the XIV century from the artist Pietro Gallo (Fig. 2C) was also analyzed with the EVA film with the aim to identify the colorant used by the artist for painting the cloak of Saint Domenicus. Although the identification and extraction of colorants is extremely complicated, the method allowed the non-invasive identification of carminic acid as reported in Table 2. The determination of carminic acid at a concentration of about 8 ng/µL suggests that the artist used the organic dye cochineal to paint the panel, probably mixed with other inorganic pigments in the form of red lake. DISCUSSION The unique ability of the EVA film here presented to harvest both macromolecules as well as small molecules from a variety of specimens and supports was demonstrated with good efficiency and without leaving any traces on the surface of the material under investigation. This last property is fundamental for using this novel extraction method on valuable material and ancient archaeological artifacts stored in museums and private collections. The EVA method differentiates completely from the first report on the use of resins for harvesting material from the surface of an original manuscript41 that could hardly be adopted as a universal harvesting technique since it could not be guaranteed that some amounts of resins could have been left behind. On the contrary, it is here demonstrated that the present EVA technology is free from such drawbacks and thus it could ultimately become the officially accepted method in such investigations. There is another important aspect that differentiates the present findings from those of Zilberstein et al.41. Only two types of beads do not represent the ideal harvesting tool. A universal capturing agent would require at least three types of beads acting simultaneously on three fundamental physico-chemical properties of any analyte, namely its positive and negative charges, as well as its hydrophobicity. We had demonstrated this time ago when studying the mechanisms of action of combinatorial peptide ligand libraries: these beads are coated with millions of hexapeptides acting on a principle of bio-affinity on the multitude of proteins present in any proteome. Among the sixteen amino acids utilized for the synthesis of these resins, eight were found to have a very strong harvesting ability, whereas the other height were mediocre capturing agents. When trying to assess the reasons of such a behaviour, we found three fundamental rules for optimal capture: those highly efficient amino acids had to have either positive charges (e.g., Lys and Arg) or negative charges (e.g. Asp and Glu) or hydrophobic regions, such as Tyr, Trp, Phe etc. Thus the best harvesting properties were linked to a three-pronged attack, namely positive and negative surface charges as well as hydrophobicity 42, 43. The EVA film here described fulfils these requirements. In particular, strong cation and strong anion exchangers are fundamental since it is known, from previous studies on the mechanism of protein capture by the combinatorial peptide ligand libraries44, that the first and most powerful docking between a bait and a protein, in an aqueous solution, is ion-ion pairing, as this is a longrange interaction and it is the strongest of all non-covalent complex formations. Once such charges, upon complex formation among proteins and their respective baits, are neutralized, further hydrophobic interactions are encouraged even in the case of more hydrophilic proteins. Obviously, in this last case, although here we have used C8 beads, it is clear that, for capture of mildly hydrophobic analytes, stronger interactors can be adopted, such as C18 or aromatic rings or any other of the hydrophobic ligand reported in the literature for HPLC. ACS Paragon Plus Environment

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The result obtained with the EVA strips allowed us to identify with high confidence different original materials on several surfaces of cultural heritage, but, like in most of the analytical surface methods, it is not possible to define with accuracy the conservation history of the object. The validation of the method for both proteins and small molecules showed excellent analytical performances and low recoveries, particularly adequate for investigations on precious ancient samples. Moreover no residues were left from the extractive film. Paintings and frescoes of old masters are composed of organic binders, colorants, glues and oils: the identification of proteins and colorants in cultural heritage objects allows a better understanding of their production techniques and ultimately of human history. Our minimally invasive method for the analysis of the organic components of ancient objects is likely to lead to surprising insights. ACKNOWLEDGMENTS The research was supported by TOTH Associazione per la ricerca scientifica ONLUS. The authors would like to thank Manital S.p.a and Rava e C s.r.l. for the accession to the historical frescoes and Dr. Baiocco from Palazzo Madama, Turin, for the accession to the wood panel from Pietro Gallo (STD16617).



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Table 1: Calibration curve results for the quantification of proteins and colorants.

SAMPLES

Linearity range (µ µg/mL)

R2

LOD (µ µg/mL)

LOQ (µ µg/mL)

RSD % conc interday (n=30)

Bovine Serum Albumin

0.005-1.000

0.9866

0.001

0.004

6.88

0.9959

0.001

0.004

7.32

Ovalbumin (Fresh egg)

(dilution from fresh egg)

Carminic acid

1.01-100.00

0.9989

0.30

1.01

8.37

Alizarin

0.93-100.00

0.9983

0.28

0.93

6.32

Indigotin

1.54-100.00

0.9927

0.46

1.54

9.71

0.003-1.000


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Table 2: Identification of proteins in frescos from the XVI century and identification of a colorant in a wood panel of the XV century: the analysis was carried out by the EVA strip extraction and LC-MS. SAMPLES

BINDER

PROTEINS (Mascot score)

Fresco I (Fresco recently discovered under the plaster) Fresco II (Fresco from the Flemish painter Paul Brill)

Casein

Beta-casein (91)

CASB_BOVIN

GPFPIIV (36 - 21) VLPVPQK (46 - 13) AVPYPQR (22 - 29)

Casein

Alpha-S1-casein (53) Beta-casein (71)

CASA1_BOVIN CASB_BOVIN

FFVAPFPEVFGK (53 - 30) GPFPIIV (25 - 24) VLPVPQK (33 - 18) AVPYPQR (26 - 26) YIPIQYVLSR (32 - 25)

SAMPLES Wood panel (from Pietro Gallo)

Accession name

PEPTIDES (ion score - identity threshold)

Kappa-casein (32)

CASK_BOV

DYE

TRANSITIONS (Q1 > Q3)

CE (V)

Concentration (ng/µL)

Carminic acid

491.0835 > 327.0516 491.0835 > 447.0940 491.0835 > 357.0622

-40 -30 -40

8.43 ± 1.40



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FIGURES Fig. 1: ESEM images of the EVA strip: the roughness of the EVA surface (a) is due to the 10-µm size SCX, SAX and C8 resins as shown on the section of the strip (b). Elemental distribution of sulphur (c), nitrogen (d) and carbon (e) on the EVA strip. Image f shows the overlapping of the sulphur (orange pixels) and carbon (blue pixels) particles.

a

20

b

11

c

d

10 20 9

40

8

60

7

80

6

5

40 60

4

80

6 100

100

3

5 120

120 4

140

3

160

2

180 50

100

150

200

250

e

0 200

1

50

100

150

200

40

60

35

80

30

100

25

120

20

140

15

160

10

180

5 50

100

150

200

250

0

250

f

45

40

200

160

1 180

200

20

2

140

50

100


150

200

25

0

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Fig. 2: Color images of the XVI century fresco from the Flemish painter Paul Brill (a), of a recently discovered fresco from Isidoro Bianchi (b) and of the wood panel (c) “San Francesco, Santi, Madonna che allatta il Bambino, San Pietro, San Paolo, Santi, la Trinità, San Giovanni Battista, Santo Vescovo, San Gerolamo” from Pietro Gallo, Italy. At the bottom of figure c there is the area (delimited in blue) sampled for the colorant analysis. In the magnification at the bottom left of image b the extractive procedure on the fresco and the 1x1 cm size EVA film are visible. Image taken by the author during the analysis at the Parella castle and at the Palazzo Madama Museum in Turin.

c

a b



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M O L E C U L E S

S M A L L P R O T E I N S

Func%onalized Film S C X C8 S A X ACS Paragon Plus Environment

Non-‐invasive Extrac%on

Page 16 of 16

LC-‐MS

Proteomics Metabolomics

Method for Noninvasive Analysis of Proteins and Small Molecules

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