Epididymal Lipocalin-Type Prostaglandin D2 Synthase: Identification Using Mass Spectrometry, Messenger RNA Localization, and Immunodetection in Mouse, Rat, Hamster, and Monkey1
Epididymal Lipocalin-Type Prostaglandin D2 Synthase: Identification Using Mass Spectrometry, Messenger RNA Localization, and Immunodetection in Mouse, Rat, Hamster, and Monkey1
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DOI：
10.1095/biolreprod66.2.524
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发表时间：
2002-02
期刊：
影响因子：
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通讯作者：
S. Fouchécourt;P. Chaurand;B. DaGue;J. Lareyre;R. Matusik;R. Caprioli;M. Orgebin‐Crist
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文献类型：
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作者：
S. Fouchécourt;P. Chaurand;B. DaGue;J. Lareyre;R. Matusik;R. Caprioli;M. Orgebin‐Crist
摘要 本研究采用二维 (2D) 凝胶电泳和质谱 (MS) 相结合的蛋白质组学方法鉴定了小鼠附睾液中的前列腺素 D2 合酶 (PGDS)。通过逆灌流收集附睾尾部液体，通过二维凝胶电泳分离蛋白质，然后在胰蛋白酶消化后进行基质辅助激光解吸电离MS分析。鉴定基于蛋白质特异性肽图以及纳米电喷雾电离 MS/MS 生成的序列信息。通过原位杂交，在头、体、尾中检测到mRNA，但在起始片段中未检测到mRNA。通过使用特异性多克隆抗体进行蛋白质印迹分析和免疫组织化学分析，PGDS 蛋白主要在体部和尾部中检测到。在尾液中，PGDS 分布在多种亚型中（pI 范围为 6.5-8.8），表明该蛋白质的一级序列经历了翻译后修饰。 N-聚糖酶消化后，分子量从 20-25 kDa 降至理论质量 18.5 kDa。在大鼠、仓鼠和食蟹猴的附睾中从头到尾也检测到了 PGDS。总之，MS 是一种强大而准确的技术，可以明确鉴定小鼠附睾 PGDS。该蛋白质 1) 存在于整个附睾中，除了起始段外，从远端头到尾部，腔内浓度逐渐增加； 2) 尾液中的主要蛋白质； 3) N-糖基化、高度多态性的蛋白质； 4）在进化过程中保守。
Abstract This study identified prostaglandin D2 synthase (PGDS) in murine epididymal fluid using a proteomic approach combining two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). The caudal epididymal fluid was collected by retroperfusion, and proteins were separated by 2D gel electrophoresis followed by matrix-assisted laser desorption ionization MS analyses after trypsin digestion. The identification was based on the protein-specific peptide map as well as on sequence information generated by nano-electrospray ionization MS/MS. By in situ hybridization, the mRNA was detected in caput, corpus, and cauda, but it was not detected in the initial segment. The PGDS protein was mostly detected in the corpus and cauda by Western blot analysis and immunohistochemistry using a specific polyclonal antibody. In caudal fluid, PGDS was distributed among several isoforms (pI range, 6.5–8.8), suggesting that this protein undergoes posttranslational modification of its primary sequence. After N-glycanase digestion, the molecular mass decreased from 20–25 to 18.5 kDa, its theoretical mass. The PGDS was also detected in the epididymis of rat, hamster, and cynomolgus monkey from the caput to the cauda. In conclusion, MS is a powerful and accurate technique that allows unambiguous identification of the murine epididymal PGDS. The protein is 1) present throughout the epididymis, except in the initial segment, with an increasing luminal concentration from distal caput to cauda; 2) a major protein in caudal fluid; 3) an N-glycosylated, highly polymorphic protein; and 4) conserved during evolution.