Faculty of Public and One Health, University of Thessaly, 43100 Karditsa, Greece.
Proteomics Research Unit, Biomedical Research Foundation of the Academy of Athens, 11527 Athens, Greece.
Int J Mol Sci. 2023 Jun 15;24(12):10160. doi: 10.3390/ijms241210160.
Based on the results of previously performed clinical studies, cathelicidin-1 has been proposed as a potential biomarker for the early diagnosis of mastitis in ewes. It has been hypothesized that the detection of unique peptides (defined as a peptide, irrespective of its length, that exists in only one protein of a proteome of interest) and core unique peptides (CUPs) (representing the shortest peptide that is unique) of cathelicidin-1 may potentially improve its identification and consequently the diagnosis of sheep mastitis. Peptides of sizes larger than those of the size of CUPs, which include consecutive or over-lapping CUPs, have been defined as 'composite core unique peptides' (CCUPs). The primary objective of the present study was the investigation of the sequence of cathelicidin-1 detected in ewes' milk in order to identify its unique peptides and core unique peptides, which would reveal potential targets for accurate detection of the protein. An additional objective was the detection of unique sequences among the tryptic digest peptides of cathelicidin-1, which would improve accuracy of identification of the protein when performing targeted MS-based proteomics. The potential uniqueness of each peptide of cathelicidin-1 was investigated using a bioinformatics tool built on a big data algorithm. A set of CUPs was created and CCUPs were also searched. Further, the unique sequences in the tryptic digest peptides of cathelicidin-1 were also detected. Finally, the 3D structure of the protein was analyzed from predicted models of proteins. In total, 59 CUPs and four CCUPs were detected in cathelicidin-1 of sheep origin. Among tryptic digest peptides, there were six peptides that were unique in that protein. After 3D structure analysis of the protein, 35 CUPs were found on the core of cathelicidin-1 of sheep origin and among them, 29 were located on amino acids in regions of the protein with 'very high' or 'confident' estimates of confidence of the structure. Ultimately, the following six CUPs: QLNEQ, NEQS, EQSSE, QSSEP, EDPD, DPDS, are proposed as potential antigenic targets for cathelicidin-1 of sheep. Moreover, another six unique peptides were detected in tryptic digests and offer novel mass tags to facilitate the detection of cathelicidin-1 during MS-based diagnostics.
基于先前进行的临床研究结果，已有人提出抗菌肽-1 可作为绵羊乳腺炎早期诊断的潜在生物标志物。人们假设，检测抗菌肽-1 的独特肽（定义为无论其长度如何，仅存在于感兴趣的蛋白质组中的一种蛋白质中的肽）和核心独特肽（CUPs）（代表最短的独特肽）可能会提高其识别能力，从而提高绵羊乳腺炎的诊断能力。大小大于 CUPs 大小的肽，包括连续或重叠的 CUPs，被定义为“复合核心独特肽”（CCUPs）。本研究的主要目的是研究在绵羊乳中检测到的抗菌肽-1 的序列，以鉴定其独特肽和核心独特肽，这将为该蛋白的准确检测揭示潜在的目标。另一个目的是检测抗菌肽-1 的胰蛋白酶消化肽中的独特序列，这将提高在进行靶向 MS 基蛋白质组学分析时该蛋白的识别准确性。使用基于大数据算法构建的生物信息学工具研究了抗菌肽-1 中每个肽的潜在独特性。创建了一组 CUPs，并搜索了 CCUPs。此外，还检测了抗菌肽-1 的胰蛋白酶消化肽中的独特序列。最后，从蛋白质的预测模型中分析了该蛋白质的 3D 结构。总共在绵羊源抗菌肽-1 中检测到 59 个 CUPs 和 4 个 CCUPs。在胰蛋白酶消化肽中，有 6 个肽是该蛋白质特有的。对绵羊源抗菌肽-1 蛋白质的 3D 结构分析后，在核心区域发现了 35 个 CUPs，其中 29 个位于蛋白质区域的氨基酸上，这些区域的结构置信度评估为“非常高”或“有信心”。最终，提出以下 6 个 CUPs：QLNEQ、NEQS、EQSSE、QSSEP、EDPD、DPDS，作为绵羊源抗菌肽-1 的潜在抗原性靶标。此外，在胰蛋白酶消化物中还检测到另外 6 个独特肽，它们提供了新的质量标签，有助于在基于 MS 的诊断中检测抗菌肽-1。
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