基质辅助激光解吸/电离飞行时间质谱法分析瘤胃脂质A

E. Sarmikasoglou, J. Vinyard, Mohamed S. Khan, Treenate Jiranantasak, A. Ravelo, R. Lobo, P. Fan, K. Jeong, A. Tuanyok, A. Faciola
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引用次数: 3

摘要

脂多糖(LPS)是革兰氏阴性菌的细胞壁成分,由三个共价连接的区域组成:o抗原、核心低聚糖和脂质A部分,其中脂质A部分具有大部分内毒活性。本研究的目的是利用基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)分离和比较全混合日粮(TMR)和放牧奶牛瘤胃脂多糖(LPS)的脂质A结构。采集2头荷兰斯坦奶牛瘤胃细菌;一组饲喂TMR(60:40,牧草精料),另一组饲喂牧草。通过将本研究奶牛的瘤胃微生物组与先前文献中的核心瘤胃微生物组进行比较,验证了每个样本的代表性。用苯酚-水萃取法提取各样品中的脂多糖,并进行超离心纯化。为了从核心和o抗原中分离脂质A,用醋酸水解纯瘤胃LPS样品。来自tmr喂养奶牛的脂质A可能表现为四酰化结构,而来自牧场喂养奶牛的脂质A可能表现为五酰化脂质A结构。两种样品均使用鲎试剂(LAL)测定法进行定量,显示出低内毒活性,与MALDI-TOF MS观察结果一致。结果表明,不同饲粮的脂质A酰化模式不同,瘤胃细菌只表达低酰化的脂质A结构,而非六酰化的脂质A,通常由大肠杆菌等细菌表达。
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Ruminal Lipid A Analysis by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Lipopolysaccharides (LPS) are cell wall components from Gram-negative bacteria and are composed of three covalently linked regions: the O-antigen, the core oligosaccharide, and the lipid A moiety, which carries most of their endotoxic activity. The objective of this study was to isolate and compare the lipid A structures from ruminal LPS derived from total mixed ration (TMR)- and pasture-fed cows, by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ruminal bacteria were collected from two rumen-cannulated Holstein cows; one fed a TMR (60:40, forage–concentrate) and the other pasture fed. The representativeness of each sample was validated by comparing the rumen microbiome from the cows in our study to the core rumen microbiome from the previous literature. Lipopolysaccharides from each respective sample were extracted with a phenol–water extraction procedure and purified via ultracentrifugation. To isolate lipid A from the core and O-antigen, pure ruminal LPS samples were hydrolyzed with acetic acid. Lipid A derived from the TMR-fed cow potentially exhibited a tetra-acylated structure, whereas lipid A derived from the pasture-fed cow potentially exhibited a penta-acylated lipid A structure. Both samples were quantified using limulus amebocyte lysate (LAL) assay and exhibited low endotoxic activity, consistent with the MALDI-TOF MS observations. Results indicate that the lipid A acylation pattern differs between diets, and that ruminal bacteria express solely under-acylated lipid A structures contrary to hexa-acylated lipid A, typically expressed by bacteria such as E. coli.
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