Miniaturisation pour l'Analyse, la Synthèse & la Protéomique (MSAP), USR CNRS 3290, and Protéomique, Modifications Post-traductionnelles et Glycobiologie, IFR 147, Université de Lille 1 Sciences et Technologies, 59655 Villeneuve d'Ascq Cedex, France.
Anal Bioanal Chem. 2011 Mar;399(9):3053-63. doi: 10.1007/s00216-010-4218-2. Epub 2010 Oct 3.
This manuscript deals with the identification of protein residues in amphorae, including particularly identification of protein species. The work described was performed on fishes, the anchovy (Engraulis encrasicolus) and bonito (Sarda sarda) species frequently found in the Mediterranean area. Based on proteomic techniques, the analytical strategy was adapted to analysis of protein residues from tiny ceramic fragments. The major difficulty was to extract proteins and limit their hydrolysis during the sample preparation; consequently, multiple soft extraction techniques were evaluated. The most valuable results were obtained using a solution containing high amounts of denaturing agents, urea and thiourea, reducing agent, dithiothreitol, and detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The analysis using nano liquid chromatography-nano electrospray ionization double quadrupole time-of-flight mass spectrometry resulted in the identification of up to 200 proteins for the anchovy and bonito species, among which 73 peptides were found to be fish-specific. Because bonito and anchovy species are not documented and fully sequenced in genomic databases, the preliminary protein identification was realized via sequence homology to other fish sequenced species. Amino acid substitutions of peptides were assigned on the basis of the interpretation of tandem mass spectrometry spectra using de novo sequencing; these peptides, not reported up to now in databases, constitute species-specific markers. The method developed was finally applied to an archaeological sample replica impregnated with a mixture of fish tissue from both species; this experiment successfully led to the identification of 17 fish proteins, including 33 fish-specific peptides. This work shows that the analytical method developed has great potential for the identification of protein species in complex archaeological samples.
本文涉及对双耳瓶中蛋白质残基的鉴定，特别是对蛋白质种类的鉴定。所描述的工作是针对在经常出现在地中海地区的鱼类，凤尾鱼（Engraulis encrasicolus）和金枪鱼（Sarda sarda）上进行的。基于蛋白质组学技术，分析策略适应了从微小陶瓷碎片中分析蛋白质残基的需要。主要的困难是提取蛋白质并在样品制备过程中限制其水解；因此，评估了多种软提取技术。使用含有高浓度变性剂、尿素和硫脲、还原剂二硫苏糖醇和去污剂 3-[(3-胆酰胺丙基)二甲氨基] -1-丙磺酸钠的溶液可获得最有价值的结果。使用纳升液相色谱-纳升电喷雾电离双四极杆飞行时间质谱分析可鉴定出多达 200 种凤尾鱼和金枪鱼物种的蛋白质，其中发现 73 种肽是鱼类特有的。由于金枪鱼和凤尾鱼物种在基因组数据库中没有被记录和完全测序，初步的蛋白质鉴定是通过与其他已测序鱼类的序列同源性来实现的。根据串联质谱图谱的从头测序解释，对肽的氨基酸取代进行了分配；这些肽在数据库中尚未报道，构成了物种特异性标志物。最后，该方法应用于浸渍有两种鱼类组织混合物的考古样本复制品；该实验成功地鉴定出 17 种鱼类蛋白质，包括 33 种鱼类特异性肽。这项工作表明，开发的分析方法在复杂的考古样本中鉴定蛋白质种类具有很大的潜力。
