Proteomic Strategies for Cultural Heritage: from Bones to Paintings☆
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Microchemical Journal 126 (2016) 341–348 Contents lists available at ScienceDirect Microchemical Journal journal homepage: www.elsevier.com/locate/microc Proteomic strategies for cultural heritage: From bones to paintings☆ Roberto Vinciguerra a, Addolorata De Chiaro a, Piero Pucci a, Gennaro Marino b, Leila Birolo a,⁎ a Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S.Angelo, Via Cinthia, Napoli, Italy b Università degli Studi ‘Suor Orsola Benincasa’, 80132 Napoli, Italy article info abstract Article history: In recent years, proteomics procedures have become increasingly popular for the characterization of proteina- Received 30 July 2015 ceous materials in ancient samples of several cultural heritage objects. The knowledge of the materials used in Received in revised form 18 December 2015 a work of art is crucial, not only to give an insight in the historical context of objects and artists, but also to analyse Accepted 18 December 2015 degradation processes taking place in aged objects and to develop appropriate conservation and/or restoration Available online 29 December 2015 treatments. However, protocols routinely applied for typical modern samples still need to be fully adapted to Keywords: take into account the low amount of proteinaceous material, the heterogeneity and the unusual physical state Proteomics of the samples, as well as the high levels of damage found in ancient samples. This paper deals with some exam- Ancient proteins ples of the adaptation of classical proteomic strategies in the analysis of ancient samples to meet the different LC-MS/MS aims in the cultural heritage field. © 2015 Elsevier B.V. All rights reserved. 1. Introduction instruments are therefore perfectly adequate to afford the minimal quantities of ancient samples. However, all the steps of the proteomic Proteomics is typically considered to be associated with the study of procedure need to be thoughtfully adapted, from the optimization of living organisms; however, its inherently multidisciplinary nature has specific protocols for sample preparation to the development of data recently led to the application of proteomic methods to oddly assorted analysis tools that can cope with ancient, damaged samples. areas ranging from forensics, food analysis, clinical medicine and even Moreover, although for merely identifying purposes, the detection of for studying the origins of life on earth; it has been proven to be an as few as two peptides is sufficient to properly pinpoint the protein, the effective tool also for the scientific analysis of artworks [1]. characterization of the modifications induced by ageing and deteriora- Proteomics for cultural heritage, i.e. the identification of proteina- tion processes requires a deeper examination of most of the protein ceous material used by artists in their masterpieces and found in archae- primary structure. ological remains, is still in its infancy, with the first paper dating back to This paper deals with the reasoning behind the choice of the steps to the early 2000 [2]. In particular, protocols routinely applied for typical be carried out in the analysis of ancient samples and with the adaptation modern samples still need to be fully adapted to take into account the of classical protocols to meet the different aims. It is not intended to low amount of proteinaceous material, the heterogeneity and the cover the whole panel of possibilities and tricks but, rather to illustrate unusual physical state of the samples, as well as the high levels of dam- the logic that might address the choice of sample treatment and data age found in ancient samples. analysis to get the most of the experiment. Although analyses in proteomics are per se invasive, modern mass spectrometry instrumentations enable the characterization of proteins 2. Material and methods with extremely high sensitivity even in crude mixtures in which the dynamic range of components abundance exceeds 1000-fold, and on Ammonium hydrogen carbonate (Ambic), Ethylenediaminetetra- very limited amount of sample, typically less than 10 μg. Modern acetic acid (EDTA); Tri(hydroxymethyl)aminomethane (TRIS), Urea, GuHCl and TPCK-treated trypsin were from Sigma; Formic acid and ☆ Selected papers presented at TECHNART 2015 Conference, Catania (Italy), April 27– Acetonitrile (ACN) were purchased from Baker. Deionized water was 30, 2015. obtained from Millipore cartridge equipment. Hydrochloric acid was ⁎ Corresponding author at: Dipartimento di Scienze Chimiche, Università di Napoli, purchased from Carlo Erba. Federico II, Napoli, Italy. Models of paint layers were prepared with milk as binders and azur- E-mail addresses: [email protected] (R. Vinciguerra), [email protected] (A. De Chiaro), [email protected] (P. Pucci), [email protected] ite (Cu3(CO3)2(OH)2), minium (Pb3O4), calcite (CaCO3), and vermilion (G. Marino), [email protected] (L. Birolo). (HgS) as pigments on glass slides and skimmed milk as control without http://dx.doi.org/10.1016/j.microc.2015.12.024 0026-265X/© 2015 Elsevier B.V. All rights reserved. 342 R. Vinciguerra et al. / Microchemical Journal 126 (2016) 341–348 pigments. Paint replicas were left to dry at RT on the bench for one A licenced version of Mascot software (www.matrixscience.com) month. Bone sample was a fragment from human bone of the first version 2.4.0. was used with trypsin as enzyme; 3, as allowed num- century A.D. ber of missed cleavage; 10 ppm MS tolerance and 0.6 Da MS/MS tol- erance; peptide charge from +2 to +3. No fixed chemical fi 2.1 Sample treatment modi cation was inserted, but possible oxidation of methionines, formation of pyroglutamic acid from glutamine residues at the N- Urea pre-treatment: 10 μL of a solution of 6 M Urea was added to terminal position of peptides, and deamidation at asparagines and fi micro-samples (ca 300–800 μg) and incubated for 10 min at RT, follow- glutamines were considered as variable modi cations [4].When fi fi ed by sonication for 20 min. Urea was then 6-fold diluted with water. collagen proteins were identi ed, a further identi cation run was EDTA pre-treatment: about 100 μL of a solution of 0.5 M EDTA was carried out, with the insertion of hydroxylation on lysine and proline fi fi fi added to the bone fragment for 10 days at RT, refreshing the solution as variable modi cations, since more con dent identi cations are every 2 days. After centrifugation for 2 min at 10,000 rpm in a benchtop commonly obtained for these proteins by taking into consideration fi microfuge, the Urea protocol described above was applied. their extensive post-translational modi cations [4].Onlyproteins HCl pre-treatment: 50 μL of 0.6 M HCl was added to the bone frag- presenting two or more peptides were considered as positively iden- fi ment and incubated at 4 °C for 4 h. After centrifugation for 2 min at ti ed. Individual ion score threshold provided by Mascot software to 10,000 rpm in a benchtop microfuge the supernatant was removed evaluate the quality of matches in MS/MS data was generally 31 for and washed with 20 μL of 10 mM Ambic. Washes were repeated for paintings and 43 for human samples. Spectra with Mascot score b four times. 100 μL of 50 mM Ambic was added and sample was left at 10 were rejected. 65 °C for 3 h. After centrifugation at 10,000 rpm for 15 min the superna- tant was removed. 3. Results and discussion GuHCl pre-treatment: 200 μL of 0.6 M HCl was added and incubated at 4 °C for 18 h. After centrifugation for 1 min at 14,000 rpm in a bench- 3.1 The minimal protocol top microfuge, and the acid-insoluble pellet washed three times with “ ” 200 μL of distilled water. The pellet was incubated at 4 °C for 72 h in a The commonly used bottom-up approach to identify proteins is buffer containing 100 mM Tris and 6 M GuHCl at pH 7.4. The sample based on the enzymatic digestion of the proteins and can be directly was then centrifuged for 1 min at 14,000 rpm in a benchtop microfuge. performed in heterogeneous phase on a sample fragment followed by The supernatant was buffer-exchanged into 10 mM Ambic using 3 K mass spectrometric analyses of the released peptides (Fig. 1). Even molecular weight cut-off Amicon Ultra, centrifugal filter unit. when the protein is embedded in a complex mixture such as that of a painting layer, few peptides released by the protease without any pre- treatment of the sample are enough to identify the protein [3], without 2.2 Protein digestion and LC-MS/MS analysis significantly affecting the sample itself. This avoid the extraction of the whole protein from the sample using harsh methods, while digestion After any pre-treatment of the sample, enzymatic digestion was can rather be carried out by depositing on the surface of a small sample carried out as in the minimally invasive proteomic analytical procedure an aqueous neutral solution containing the enzyme that directly trims fl fi described by Leo et al. [3].Brie y, trypsin was added to a nal concentra- protruding peptides. The solution will then be gently removed and, μ – μ tion of 10 ng/ L to micro-samples (ca 300 800 g) as directly suspended once released, peptides can be analysed by mass spectrometric methods μ in 50 L of Ambic 10 mM. After incubation at 37 °C for 16 h, the superna- such as MALDI-TOF and LC-MS/MS, and database searches by bioinfor- tants were recovered by centrifugation at 10,000 rpm, and the peptide matics tools such as Mascot (www.matrixscience.com) allow protein fi μ mixture was ltered on 0.22 m PVDF membrane (Millipore), concen- identification.