Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing Peptide Mass Fingerprinting and LC-Based Proteomics Methods.
Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing Peptide Mass Fingerprinting and LC-Based Proteomics Methods.
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DOI：
10.3390/ijms17040445
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发表时间：
2016-03-24
影响因子：
5.6
通讯作者：
Buckley M
中科院分区：
文献类型：
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作者：
Buckley M
胶原蛋白是动物界中最普遍的蛋白质之一，也是骨、皮肤等细胞外组织中的主要蛋白质，在这些组织中它主要充当支架的作用。人们对它进行了广泛的科学研究，不仅作为再生医学的生物医学材料，而且还作为人类和牲畜的食物来源。由于胶原蛋白的长期稳定性以及其在骨骼中的丰度，它已被提议作为生物标记物的来源，不仅用于热和压力处理的动物饲料，而且还用于古代考古和古生物标本的鉴定，通常通过多肽质量指纹图谱(PMF)以及基于深度液相色谱(LC)的串联质谱学方法进行。通过对牛、羊和猪这三种最常见的家养动物的分析，本研究调查了每种方法相对于另一种方法的优势，调查了胶原蛋白分子已知功能特性的序列变异位置。结果表明，之前通过PMF分析鉴定的物种生物标记物并不是这些组织中存在的最可变的1型胶原肽，后者可以用基于LC的方法来检测。然而，很明显，整个分子中胶原蛋白的高度重复序列基序，加上羟化位点和相对丰度水平的可变性，可以导致使用这些基于LC的方法进行高分假阳性多肽匹配。此外，与α1(I)链相比，更大的α2(I)链序列变异似乎不是任何特定功能特性所特有的，这意味着对序列变异的链内功能约束不如链间约束那么大。然而，尽管一些最易变的多肽只在基于LC的方法中观察到，但在公开可用的胶原序列的范围得到改善之前，PMF方法的简单性和观察到的肽序列变化的合适范围使其成为仅在需要时才通过基于LC的方法进行进一步分析之前的初始分类鉴定的理想方法。
Collagen is one of the most ubiquitous proteins in the animal kingdom and the dominant protein in extracellular tissues such as bone, skin and other connective tissues in which it acts primarily as a supporting scaffold. It has been widely investigated scientifically, not only as a biomedical material for regenerative medicine, but also for its role as a food source for both humans and livestock. Due to the long-term stability of collagen, as well as its abundance in bone, it has been proposed as a source of biomarkers for species identification not only for heat- and pressure-rendered animal feed but also in ancient archaeological and palaeontological specimens, typically carried out by peptide mass fingerprinting (PMF) as well as in-depth liquid chromatography (LC)-based tandem mass spectrometric methods. Through the analysis of the three most common domesticates species, cow, sheep, and pig, this research investigates the advantages of each approach over the other, investigating sites of sequence variation with known functional properties of the collagen molecule. Results indicate that the previously identified species biomarkers through PMF analysis are not among the most variable type 1 collagen peptides present in these tissues, the latter of which can be detected by LC-based methods. However, it is clear that the highly repetitive sequence motif of collagen throughout the molecule, combined with the variability of the sites and relative abundance levels of hydroxylation, can result in high scoring false positive peptide matches using these LC-based methods. Additionally, the greater alpha 2(I) chain sequence variation, in comparison to the alpha 1(I) chain, did not appear to be specific to any particular functional properties, implying that intra-chain functional constraints on sequence variation are not as great as inter-chain constraints. However, although some of the most variable peptides were only observed in LC-based methods, until the range of publicly available collagen sequences improves, the simplicity of the PMF approach and suitable range of peptide sequence variation observed makes it the ideal method for initial taxonomic identification prior to further analysis by LC-based methods only when required.