Solazzo Caroline, Fitzhugh William W, Rolando Christian, Tokarski Caroline
Chimie Organique et Macromoléculaire, UMR CNRS 8009, and Protéomique, Modifications Post-traductionnelles et Glycobiologie, IFR 147, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France.
Anal Chem. 2008 Jun 15;80(12):4590-7. doi: 10.1021/ac800515v.
We demonstrate here the possibility of identifying proteins trapped in few milligrams of the clay matrix of a 1200-1400 AD Iñupiat potsherd fragment from Point Barrow, Alaska, by a dedicated proteomics approach. The four main steps of a proteomics analysis, (i) protein extraction from biological samples, (ii) protein hydrolysis using a hydrolase enzyme, (iii) nanoLC, nanoESI MS, and MS/MS analysis of the generated peptides, and (iv) protein identification using protein databank proceeded from genomic data, have been optimized for archeological remains. Briefly our procedure starts by grinding the potsherds, extraction with 1% trifluoroacetic acid, digestion with excess of trypsin, nanoLC, nanoESI FT-ICR analysis, and data mining by homology search. The developed conditions were evaluated on protein extracts from remains obtained by heated muscle tissues and blubbers of different seal and whale species, these samples representing the main diet sources of the Eskimo population. Most of the proteins were identified by sequence homology to other species due to the lack of cetacean and pinniped proteins in the databanks. More interestingly, two proteins, myoglobin and hemoglobin, respectively, identified in muscle tissue samples and blubber samples highlight several specific peptides of cetacean and pinniped species; these peptides are significant to prove the presence of these marine species in the analyzed samples. Based on the developed methodology and on protein identification results obtained from the heated seal/whale muscle tissues and blubbers, the analysis of the clay matrix of a 1200-1400 AD Iñupiat potsherd fragment from Point Barrow was investigated. The described method succeeds in identifying four peptides corresponding to the harbor seal myoglobin (species Phoca vitulina) with a measured mass accuracy better than 1 ppm (MS and MS/MS experiments) including one specific peptide of the cetacean and pinniped species and one specific peptide of the seal species. These results highlight, for the first time, a methodology able to identify proteins from a few milligrams of archeological potsherd buried for years; the obtained results confirm the presence of a seal muscle tissue protein in this Punuk potsherd.
我们在此展示了通过一种专门的蛋白质组学方法，从阿拉斯加巴罗角公元1200 - 1400年伊努皮亚特陶罐碎片几毫克的黏土基质中鉴定蛋白质的可能性。蛋白质组学分析的四个主要步骤，即（i）从生物样品中提取蛋白质，（ii）使用水解酶进行蛋白质水解，（iii）对生成的肽段进行纳升液相色谱、纳升电喷雾质谱和串联质谱分析，以及（iv）使用源自基因组数据的蛋白质数据库进行蛋白质鉴定，已针对考古遗迹进行了优化。简而言之，我们的流程始于研磨陶罐碎片，用1%的三氟乙酸提取，用过量胰蛋白酶消化，纳升液相色谱、纳升电喷雾傅里叶变换离子回旋共振分析，以及通过同源搜索进行数据挖掘。所开发的条件在取自不同海豹和鲸鱼物种的加热肌肉组织和鲸脂的遗迹蛋白质提取物上进行了评估，这些样品代表了爱斯基摩人群的主要饮食来源。由于数据库中缺乏鲸类和鳍足类动物的蛋白质，大多数蛋白质是通过与其他物种的序列同源性鉴定出来的。更有趣的是，分别在肌肉组织样品和鲸脂样品中鉴定出的两种蛋白质，即肌红蛋白和血红蛋白，突出了鲸类和鳍足类物种的几个特定肽段；这些肽段对于证明分析样品中这些海洋物种的存在具有重要意义。基于所开发的方法以及从加热的海豹/鲸鱼肌肉组织和鲸脂中获得的蛋白质鉴定结果，对阿拉斯加巴罗角公元1200 - 1400年伊努皮亚特陶罐碎片的黏土基质进行了分析。所描述的方法成功鉴定出了四个与港海豹肌红蛋白（物种为Phoca vitulina）相对应的肽段，在质谱和串联质谱实验中测得的质量准确度优于1 ppm，其中包括一个鲸类和鳍足类物种的特定肽段以及一个海豹物种的特定肽段。这些结果首次突出了一种能够从埋藏多年的几毫克考古陶罐碎片中鉴定蛋白质的方法；所获得的结果证实了该普努克陶罐碎片中存在海豹肌肉组织蛋白。
