National Institute of Standards and Technology, Analytical Chemistry Division, Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, SC 29412, USA.
Metallomics. 2012 Jun;4(6):533-8. doi: 10.1039/c2mt20059g. Epub 2012 May 3.
Selenoprotein P (SePP) is a unique selenium-containing protein responsible for the transport and distribution of the essential trace element selenium (Se) through the human body with the concentration of SePP in human blood representing the most useful marker of Se nutritional status. Although SePP has been extensively studied, the structure of SePP in human plasma remains unresolved. Two potential isoforms of SePP have been identified by Western blot analyses distinguished principally by differences in migration (51 kDa and 61 kDa). The biological relevance of the smaller isoform has been called into question by several studies reporting only one major SePP form (69 kDa) suggesting that the shorter 51 kDa is an artifact of protease activity during the SePP purification process. A deficiency of these Western blot analyses is that no information can be gleaned regarding the Se content of the potential isoforms. This study reports a characterization of SePP isoforms in a human plasma Standard Reference Material representative of a healthy US population. Following immunoprecipitation, three SePP isoforms were unequivocally identified at 45 kDa, 49 kDa and 57 kDa (termed as SePP45, SePP49 and SePP57) by LC-MS/MS analyses from a spectral searching approach. Selenium (Se) was detected by gel electrophoresis LA-ICP-MS in SePP49 and SePP57 which was confirmed by the identification of three selenopeptides covering the SePP sequence from residues 312-346 by LC-MS/MS analyses utilizing a sequence searching approach. Conversely, neither Se nor peptides covering SePP sequence from residues 306-346 was identified in SePP45 which suggests that SePP45 is a truncated isoform transcriptionally terminated at the 2nd in-frame UGA codon thereby terminating the protein with the Ser residue at position 299. An additional band at 23 kDa was found to contain Se but no peptides of SePP. Instead, glutathione peroxidase 3 (GPx3) was unequivocally identified within the band presumably being co-immunoprecipitated with the SePP providing preliminary evidence that SePP and GPx3 interaction may take place in vivo.
硒蛋白 P（SePP）是一种独特的含硒蛋白，负责通过人体运输和分配必需的微量元素硒，人体血液中的 SePP 浓度是硒营养状况最有用的标志物。尽管 SePP 已经被广泛研究，但人类血浆中的 SePP 结构仍未得到解决。通过 Western blot 分析鉴定出两种潜在的 SePP 同工型，主要区别在于迁移率不同（51 kDa 和 61 kDa）。几项研究报告仅有一种主要的 SePP 形式（69 kDa），从而对较小同工型的生物学相关性提出了质疑，这表明较短的 51 kDa 是 SePP 纯化过程中蛋白酶活性的一种假象。这些 Western blot 分析的一个缺点是，无法获得潜在同工型的硒含量信息。本研究报告了一种针对代表美国健康人群的人类血浆标准参考物质中 SePP 同工型的特征描述。通过免疫沉淀后，通过 LC-MS/MS 分析从谱搜索方法中明确鉴定出 45 kDa、49 kDa 和 57 kDa 三种 SePP 同工型（分别称为 SePP45、SePP49 和 SePP57）。通过凝胶电泳 LA-ICP-MS 在 SePP49 和 SePP57 中检测到硒（Se），通过 LC-MS/MS 分析利用序列搜索方法鉴定覆盖 SePP 序列从残基 312-346 的三个硒肽证实了这一点。相反，在 SePP45 中既未检测到 Se 也未检测到覆盖 SePP 序列从残基 306-346 的肽，这表明 SePP45 是一种转录终止于第二个框内 UGA 密码子的截断同工型，从而使蛋白质在位置 299 处终止为丝氨酸残基。在 23 kDa 处发现一个带含有 Se，但没有 SePP 的肽。相反，谷胱甘肽过氧化物酶 3（GPx3）在该带中被明确鉴定，推测其与 SePP 共同免疫沉淀，这提供了初步证据表明 SePP 和 GPx3 之间的相互作用可能在体内发生。
