A novel strategy based on in situ dual-enzyme digestion of paint layer proteinaceous binders is introduced for faster and more confident identification, resulting in a bottom-up proteomics approach by MALDI-TOF mass spectrometry (MS). In situ sampling/extraction of proteinaceous binders using small pieces of a hydrophilic gel, previously loaded with trypsin and chymotrypsin proteolytic enzymes, was successfully exploited. Along with minimal invasiveness, the synergy of both enzymes was very useful to increase the number of annotated peptide peaks with their corresponding amino acid sequence by database search and subsequent MALDI-TOF/TOF analysis. The protocol was initially aimed at enhancing the identification of egg-based binders and then validated on fresh and aged model pictorial layers; an increased protein coverage was significantly attained regardless of the used painting binders. Optical microscope images and spectrophotocolorimetry analysis evidenced that the painting layers were not damaged or altered because of contact/sampling without leaving hydrogel residues. The proposed protocol was successfully applied on a painted altarpiece “Assumption of the Virgin” dated to the XVI century and on an angel statue of the Nativity crib dated to the XII century, both from Altamura’s Cathedral (Apulia, Italy). The occurrence of various protein binders of animal origin was easily and reliably ascertained.

中文翻译：

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蛋白质粘合剂的双酶消化原位水凝胶萃取：艺术品可靠质谱研究的关键。

引入了一种基于原位双酶消化漆层蛋白质粘合剂的新策略，以更快，更可靠地进行鉴定，从而通过MALDI-TOF质谱（MS）实现了自下而上的蛋白质组学方法。原位已成功开发了使用小块亲水凝胶（以前装有胰蛋白酶和胰凝乳蛋白酶蛋白水解酶）对蛋白质粘合剂进行采样/提取的方法。除了极小的侵袭性，两种酶的协同作用对于通过数据库搜索和随后的MALDI-TOF / TOF分析增加带注释的肽峰及其相应氨基酸序列的数量非常有用。该协议最初旨在增强对蛋基粘合剂的识别，然后在新鲜的和陈旧的模型图片层上进行验证。无论使用哪种绘画粘合剂，均可显着提高蛋白质的覆盖率。光学显微镜图像和分光光度法分析表明，由于接触/取样而没有留下水凝胶残留物，因此涂装层没有损坏或改变。拟议中的协议已成功地应用于阿尔塔穆拉大教堂（意大利普利亚）的十六世纪的彩绘祭坛“圣母升天”和十二世纪的耶稣诞生婴儿床天使雕像。可以容易且可靠地确定各种动物来源的蛋白结合剂的出现。