Materials and reagents
Analytical high-performance liquid chromatography (HPLC) solvents, including methanol, ethanol, chloroform, hexane, and dichloromethane were obtained from JT Baker (Deventer, Netherlands). Hydrochloric acid 37% was purchased from Carlo Erba (Cornaredo, Italy). Ammonium bicarbonate was purchased from Merck (Merck KGaA, Darmstadt, Germany). Amino acid calibration standards in 0.1 mol/L HCl, borate buffer 0.4 mol/L in water (pH 10.2), and 10 mg/mL o-phthalaldehyde- 3-mercaptopropionic acid (OPA-3-MPA) reagent in borate buffer (0.4 mol/L) were obtained from Agilent Technologies GmbH & Co.KG (Waldbronn, Germany). Dithiothreitol (DTT) was purchased from Sigma Aldrich (St. Louis, MO, USA). The pentadecanoic acid (C15:0, Sigma, St. Louis, MO, USA) was dissolved in ethanol at a final concentration of 0.2 mg/mL. Boron trifluoride/methanol (10%, w/w) was purchased from Supelco (Bellefonte, PA, USA).
Wall painting samples and sampling step preparation
Five sporadic wall painting samples were selected from a large collection of archaeological ruins stored in Pompeii’s house of Golden Bracelet, Insula Occidentalis. The specimens (about 5 × 3 × 2 cm, see Fig. 1) were carefully handled to prevent further contamination and gently cleaned with a soft brush and wet bibula paper to remove dust deposit. All samples surface did not show the presence of any evident patina not belonging to the painting layer. The first sample was mostly of a grey color with brown decoration, the second contained intense red and green coloration, the third showed green decoration on a yellowish background, the fourth showed light green violet and orange colors, and the fifth was made of weak red color. For chemical analyses, amounts of wall painting powder ranging between 60 and 130 mg were scraped with a new disposable scalpel from sample surface whereas amounts between 50 and 130 mg were scraped from the below corresponding remaining calcite layer. Powders were suspended in distilled water up to a concentration of 200 mg/mL and then mechanically pulverized on ice for 5 min at half maximum speed by using an Ultra Turrax T25 Digital Homogenizer (IKA®-Werke GmbH & Co. KG, Staufen, Germany). The extraction of polar and nonpolar compounds was carried out according to the method of the Standard Metabolic Reporting Structures working group [8].
FT-IR spectroscopy
Samples (200 mg/mL) were diluted at different concentration of 10, 5 and 2.5 μg/μL. 3 μL of each suspension (corresponding to 30, 15 and 7.5 μg) was layered on 3 mm ZnS window, dried under a white lamp (60 watts) and analyzed with a Nicolet 5700 equipped with a microscope ContinuμM (Thermo Fisher Scientific, Waltham, MA). For each sample, 3 spectra (200 acquisitions) in transmission mode were collected, with a sensitivity of eight, and the microscope focusing windows set at 100 × 100 μm. Peak areas were then evaluated by using the Omnic software (Thermo Fisher Scientific).
Evaluation of oxalate was performed by FT-IR in the spectral region between 690 and 890 cm−1, by measuring for each sample the peak area at around 780 cm−1. Using the OriginPro 7.5 software spectra deconvolution and curve fitting was performed. Peak assignment was evaluated on the basis of the data library [9].
Amino acid and protein analysis
An aliquot of each wall painting samples corresponding to 10 mg of powder was dried under nitrogen and suspended in 100 μL of 50 mM ammonium bicarbonate, sonicated for 10 min, vortexed and centrifuged at 14,000 rpm for 10 min. The supernatants were collected and subjected to pre-column derivatization in the needle of the autosampler with OPA-3-MPA, and injected in the HPLC system as previously reported [10]. Briefly, each sample (25 μL) was transferred into a conical vial insert for pre-column derivatization, and the amino acid concentration was determined using the calibration curve. Amino acids were identified and quantified by comparison of their retention time and absorption ratio with those of authentic compounds in the calibration solution containing 25 amino acids (Asp, Glu, Asa, Asn, Ser, Glx, His, Gly, Thr, Cit, Arg, Ala, Tau, Tyr, Val, Met, Trp, Phe, Ile, Orn, Leu, Lys, Oxo-Pro, Sarc, Pro) and norvaline as internal standard. The analyses were performed using an Agilent Technologies 1200 Series LC System (Agilent, Santa Clara, CA) equipped with a binary pump delivery system, an autosampler to automate the pre-column derivatization and injection procedure, a heated column compartment, and a programmable fluorescence detector. All of the equipment was controlled by Agilent ChemStation software. An Agilent Zorbax Eclipse XDB-C18 analytical column (5 μm, 4.6 × 150 mm), was used in parallel to an Agilent Eclipse XDB-C18 analytical Guard column (5 μm, 4.6 × 12.5 mm) for chromatographic separations.
For the analysis of proteins, 50 μL of the ammonium bicarbonate supernatant was also incubated in 5.8 mM dithiothreitol (DTT) for 5 min at 95 °C for disulfide bridges reduction and analyzed according to the procedure described by Chambery et al. [11].
Lipid analysis
An aliquot of each wall painting samples corresponding to 50 mg of powder was dried under nitrogen and C15:0 (10 μg) was added to each powder as an internal standard. Methanol (800 μL) and water (170 μL) were added and the samples were vortex for 3 min. Then, chloroform (400 μL) was added, and the samples were incubated on ice for 10 min. Finally, chloroform (400 μL) and water (400 μL) were added to the samples and, after vortex mixing for 3 min, the samples were centrifuged at 3000 rpm for 30 min. The lower layer (lipophilic phase) was filtered by glass microfiber filter, dried under a stream of nitrogen, and re-suspended in 500 μL of BF3/methanol (10%, w/w). Trans-esterification of complex lipids was performed at 60 °C for 20 min. Then, the sample was mixed with 1 mL of distilled water and the methylated fatty acids were extracted two times with 1 mL of hexane. The samples were dried under a gentle nitrogen stream and the residue was dissolved in 100 μL of CH2Cl2. Aliquots (1 μL) of the samples were analyzed by GC-FID (HP-5890, Agilent) and GC–MS (GC 8000/MD800, Fisons Instruments) controlled by a workstation equipped with MassLab 3.4 software [7].