Globe Institute, University of Copenhagen, Copenhagen, Denmark.
Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.
J Proteomics. 2024 Apr 30;298:105143. doi: 10.1016/j.jprot.2024.105143. Epub 2024 Feb 27.
An increasing number of studies utilise the recovery of ancient skeletal proteomes for phylogenetic and evolutionary analysis. Although these studies manage to extract and analyse ancient peptides, the recovered proteomes are generally small in size and with low protein sequence coverage. We expand on previous observations which have shown that the parallel digestion and analysis of Pleistocene skeletal proteomes increases overall proteome size and protein sequence coverage. Furthermore, we demonstrate that the consecutive digestion of a skeletal proteome using two proteases, particularly the combination of Glu-C or chymotrypsin followed by trypsin digestion, enables the recovery of alternative proteome components not reachable through trypsin digestion alone. The proteomes preserved in Pleistocene skeletal specimens are larger than previously anticipated, but unlocking this protein sequence information requires adaptation of extraction and protein digestion protocols. The sequential utilisation of several proteases is, in this regard, a promising avenue for the study of highly degraded but unique hominin proteomes for phylogenetic purposes. SIGNIFICANCE: Palaeoproteomic analysis of archaeological materials, such as hominin skeletal elements, show great promise in studying past organisms and evolutionary relationships. However, as most proteomic methods are inherently destructive, it is essential to aim to recover as much information as possible from every sample. Currently, digestion with trypsin is the standard approach in most palaeoproteomic studies. We find that parallel or consecutive digestion with multiple proteases can improve proteome size and coverage for both Holocene and Pleistocene bone specimens. This allows for recovery of more proteomic data from a sample and maximises the chance of recovering phylogenetically relevant information.
越来越多的研究利用古代骨骼蛋白质组的恢复进行系统发育和进化分析。虽然这些研究设法提取和分析了古代肽，但恢复的蛋白质组通常规模较小，蛋白质序列覆盖率较低。我们扩展了以前的观察结果，即平行消化和分析更新世骨骼蛋白质组可以增加总体蛋白质组大小和蛋白质序列覆盖率。此外，我们证明，使用两种蛋白酶（特别是 Glu-C 或胰凝乳蛋白酶消化后紧接着使用胰蛋白酶消化）连续消化骨骼蛋白质组，可以恢复通过单独使用胰蛋白酶消化无法获得的替代蛋白质组成分。保存在更新世骨骼标本中的蛋白质组比以前预期的要大，但要解锁这些蛋白质序列信息，需要适应提取和蛋白质消化方案。在这方面，连续使用几种蛋白酶是研究高度降解但独特的人类蛋白质组以进行系统发育目的的有前途的途径。 意义：对考古材料（如人类骨骼元素）的古蛋白质组分析在研究过去的生物和进化关系方面显示出巨大的潜力。然而，由于大多数蛋白质组学方法本质上是具有破坏性的，因此从每个样本中尽可能多地恢复信息是至关重要的。目前，在大多数古蛋白质组学研究中，使用胰蛋白酶消化是标准方法。我们发现，使用多种蛋白酶进行平行或连续消化可以提高全新世和更新世骨骼标本的蛋白质组大小和覆盖率。这允许从一个样本中恢复更多的蛋白质组数据，并最大限度地提高恢复与系统发育相关信息的机会。
