Bray Fabrice, Fabrizi Isabelle, Flament Stéphanie, Locht Jean-Luc, Antoine Pierre, Auguste Patrick, Rolando Christian
Univ. Lille, CNRS, UAR 3290─MSAP - Miniaturisation pour La Synthèse, L'Analyse et La Protéomique, Lille F-59000, France.
Inrap Hauts-de-France, 32, avenue de l'Étoile-du-Sud, Glisy 80440, France.
Anal Chem. 2023 May 16;95(19):7422-7432. doi: 10.1021/acs.analchem.2c03301. Epub 2023 May 2.
Peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry allows the identification of bone species based on their type I collagen sequence. In the archaeological or paleontological field, PMF is known as zooarchaeology mass spectrometry (ZooMS) and is widely implemented to find markers for most species, including the extinct ones. In addition to the identification of bone species, ZooMS enables dating estimation by measuring the deamidation value of specific peptides. Herein, we report several enhancements to the classical ZooMS technique, which reduces to 10-fold the required bone sample amount (down to the milligram scale) and achieves robust deamidation value calculation in a high-throughput manner. These improvements rely on a 96-well plate samples preparation, a careful optimization of collagen extraction and digestion to avoid spurious post-translational modification production, and PMF at high resolution using matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance (MALDI-FTICR) analysis. This method was applied to the identification of a hundred bones of herbivores from the Middle Paleolithic site of Caours (Somme, France) well dated from the Eemian Last Interglacial climatic optimum. The method gave reliable species identification to bones already identified by their osteomorphology, as well as to more challenging samples consisting of small or burned bone fragments. Deamidation values of bones originating from the same geological layers have a low standard deviation. The method can be applied to archaeological bone remains and offers a robust capacity to identify traditionally unidentifiable bone fragments, thus increasing the number of identified specimens and providing invaluable information in specific contexts.
使用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)的肽质量指纹图谱(PMF)技术可根据I型胶原蛋白序列鉴定骨骼所属物种。在考古学或古生物学领域,PMF被称为动物考古质谱法(ZooMS),已被广泛应用于寻找大多数物种(包括已灭绝物种)的标记物。除了鉴定骨骼物种外,ZooMS还能通过测量特定肽段的脱酰胺值来估计年代。在此,我们报告了对经典ZooMS技术的多项改进,这些改进将所需的骨骼样本量减少到原来的十分之一(低至毫克级别),并以高通量方式实现了可靠的脱酰胺值计算。这些改进依赖于96孔板样本制备、对胶原蛋白提取和消化的精心优化以避免产生虚假的翻译后修饰,以及使用基质辅助激光解吸电离傅里叶变换离子回旋共振(MALDI-FTICR)分析进行高分辨率的PMF分析。该方法应用于鉴定来自法国索姆省考尔的旧石器时代中期遗址的一百块食草动物骨骼,这些骨骼来自艾姆间冰期气候适宜期,年代测定准确。该方法不仅能可靠地鉴定出已通过骨形态学鉴定的骨骼物种,还能鉴定更具挑战性的小骨碎片或烧焦骨碎片样本。来自同一地质层的骨骼的脱酰胺值具有较低的标准差。该方法可应用于考古骨骼遗骸,具有强大的能力来鉴定传统上难以鉴定的骨碎片,从而增加已鉴定标本的数量,并在特定情况下提供宝贵信息。