Abstract：
BACKGROUND: Tick hemolymph bathes internal organs, acts as an exchange medium for nutrients and cellular metabolites, and offers protection against pathogens. Hemolymph is abundant in proteins. However, there has been limited integrated protein analysis in tick hemolymph thus far. Moreover, there are difficulties in differentiating tick-derived proteins from the host source. The aim of this study was to profile the tick/host protein components in the hemolymph of Haemaphysalis flava.
METHODS: Hemolymph from adult engorged H. flava females was collected by leg amputation from the Erinaceus europaeus host. Hemolymph proteins were extracted by a filter-aided sample preparation protocol, digested by trypsin, and assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS raw data were searched against the UniProt Erinaceidae database and H. flava protein database for host- and tick-derived protein identification. Protein abundance was further quantified by intensity-based absolute quantification (iBAQ).
RESULTS: Proteins extracted from hemolymph unevenly varied in size with intense bands between 100 and 130 kDa. In total, 312 proteins were identified in the present study. Therein 40 proteins were identified to be host-derived proteins, of which 18 were high-confidence proteins. Top 10 abundant host-derived proteins included hemoglobin subunit-α and subunit-β, albumin, serotransferrin-like, ubiquitin-like, haptoglobin, α-1-antitrypsin-like protein, histone H2B, apolipoprotein A-I, and C3-β. In contrast, 169 were high-confidence tick-derived proteins. These proteins were classified into six categories based on reported functions in ticks, i.e., enzymes, enzyme inhibitors, transporters, immune-related proteins, muscle proteins, and heat shock proteins. The abundance of Vg, microplusin and α-2-macroglobulin was the highest among tick-derived proteins as indicated by iBAQ.
CONCLUSIONS: Numerous tick- and host-derived proteins were identified in hemolymph. The protein profile of H. flava hemolymph revealed a sophisticated protein system in the physiological processes of anticoagulation, digestion of blood meal, and innate immunity. More investigations are needed to characterize tick-derived proteins in hemolymph.
METHODS: Hemolymph from adult engorged H. flava females was collected by leg amputation from the Erinaceus europaeus host. Hemolymph proteins were extracted by a filter-aided sample preparation protocol, digested by trypsin, and assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS raw data were searched against the UniProt Erinaceidae database and H. flava protein database for host- and tick-derived protein identification. Protein abundance was further quantified by intensity-based absolute quantification (iBAQ).
RESULTS: Proteins extracted from hemolymph unevenly varied in size with intense bands between 100 and 130 kDa. In total, 312 proteins were identified in the present study. Therein 40 proteins were identified to be host-derived proteins, of which 18 were high-confidence proteins. Top 10 abundant host-derived proteins included hemoglobin subunit-α and subunit-β, albumin, serotransferrin-like, ubiquitin-like, haptoglobin, α-1-antitrypsin-like protein, histone H2B, apolipoprotein A-I, and C3-β. In contrast, 169 were high-confidence tick-derived proteins. These proteins were classified into six categories based on reported functions in ticks, i.e., enzymes, enzyme inhibitors, transporters, immune-related proteins, muscle proteins, and heat shock proteins. The abundance of Vg, microplusin and α-2-macroglobulin was the highest among tick-derived proteins as indicated by iBAQ.
CONCLUSIONS: Numerous tick- and host-derived proteins were identified in hemolymph. The protein profile of H. flava hemolymph revealed a sophisticated protein system in the physiological processes of anticoagulation, digestion of blood meal, and innate immunity. More investigations are needed to characterize tick-derived proteins in hemolymph.
摘要：
背景：滴答血淋巴浴内脏器官，作为营养物质和细胞代谢产物的交换介质，并提供对病原体的保护。血淋巴富含蛋白质。然而,到目前为止，在蜱血淋巴中的整合蛋白分析有限。此外，从宿主来源区分蜱源蛋白存在困难。这项研究的目的是对黄藻血淋巴中的tick/宿主蛋白成分进行分析。
方法：通过截肢从Erinaceuseuropaeus宿主中收集成年吞食H.fla雌性的血淋巴。通过过滤辅助样品制备方案提取血淋巴蛋白，被胰蛋白酶消化，并通过液相色谱-串联质谱(LC-MS/MS)测定。针对UniProtErinaceidae数据库和H.flava蛋白数据库搜索MS原始数据，以进行宿主和壁虱来源的蛋白鉴定。通过基于强度的绝对定量(iBAQ)进一步定量蛋白质丰度。
结果：从血淋巴中提取的蛋白质大小不均匀地变化，在100至130kDa之间具有强烈的条带。总的来说,在本研究中鉴定出312种蛋白质。其中40种蛋白质被鉴定为宿主来源的蛋白质，其中18是高置信度蛋白。前10个丰富的宿主衍生蛋白包括血红蛋白亚基-α和亚基-β，白蛋白,类血清转铁蛋白,泛素样，触珠蛋白，α-1-抗胰蛋白酶样蛋白,组蛋白H2B,载脂蛋白A-I,和C3-β。相比之下,169个是高置信度蜱衍生的蛋白质。这些蛋白质根据报告的壁虱功能分为六类，即，酶，酶抑制剂，运输商,免疫相关蛋白，肌肉蛋白质，和热休克蛋白。丰富的Vg，如iBAQ所示，microplusin和α-2-巨球蛋白在tick衍生的蛋白质中最高。
结论：在血淋巴中发现了许多蜱和宿主来源的蛋白质。黄曲霉血淋巴的蛋白质谱揭示了抗凝生理过程中复杂的蛋白质系统，消化血粉，和先天免疫。需要更多的研究来表征血淋巴中的蜱衍生蛋白。
方法：通过截肢从Erinaceuseuropaeus宿主中收集成年吞食H.fla雌性的血淋巴。通过过滤辅助样品制备方案提取血淋巴蛋白，被胰蛋白酶消化，并通过液相色谱-串联质谱(LC-MS/MS)测定。针对UniProtErinaceidae数据库和H.flava蛋白数据库搜索MS原始数据，以进行宿主和壁虱来源的蛋白鉴定。通过基于强度的绝对定量(iBAQ)进一步定量蛋白质丰度。
结果：从血淋巴中提取的蛋白质大小不均匀地变化，在100至130kDa之间具有强烈的条带。总的来说,在本研究中鉴定出312种蛋白质。其中40种蛋白质被鉴定为宿主来源的蛋白质，其中18是高置信度蛋白。前10个丰富的宿主衍生蛋白包括血红蛋白亚基-α和亚基-β，白蛋白,类血清转铁蛋白,泛素样，触珠蛋白，α-1-抗胰蛋白酶样蛋白,组蛋白H2B,载脂蛋白A-I,和C3-β。相比之下,169个是高置信度蜱衍生的蛋白质。这些蛋白质根据报告的壁虱功能分为六类，即，酶，酶抑制剂，运输商,免疫相关蛋白，肌肉蛋白质，和热休克蛋白。丰富的Vg，如iBAQ所示，microplusin和α-2-巨球蛋白在tick衍生的蛋白质中最高。
结论：在血淋巴中发现了许多蜱和宿主来源的蛋白质。黄曲霉血淋巴的蛋白质谱揭示了抗凝生理过程中复杂的蛋白质系统，消化血粉，和先天免疫。需要更多的研究来表征血淋巴中的蜱衍生蛋白。
