Department of Veterinary and Biomedical Sciences and The Penn State Cancer Institute, The Pennsylvania State University, 115 Henning Building, University Park, PA, 16802, USA.
Department of Pathology, Proteomics Research Core Facility, Medical School, University of Michigan, Ann Arbor, MI, USA.
J Am Soc Mass Spectrom. 2019 Jul;30(7):1276-1283. doi: 10.1007/s13361-019-02192-9. Epub 2019 Apr 9.
Selenium (Se) functions as a cellular redox gatekeeper through its incorporation into proteins as the 21st amino acid, selenocysteine (Sec). Supplementation of macrophages with exogenous Se (as sodium selenite) downregulates inflammation and intracellular oxidative stress by effectively restoring redox homeostasis upon challenge with bacterial endotoxin lipopolysaccharide (LPS). Here, we examined the use of a standard Tandem Mass Tag (TMT)-labeling mass spectrometry-based proteomic workflow to quantitate and examine temporal regulation of selenoproteins in such inflamed cells. Se-deficient murine primary bone marrow-derived macrophages (BMDMs) exposed to LPS in the presence or absence of selenite treatment for various time periods (0-20 h) were used to analyze the selenoproteome expression using isobaric labeling and shotgun proteomic workflow. To overcome the challenge of identification of Sec peptides, we used the identification of non-Sec containing peptides downstream of Sec as a reliable evidence of ribosome readthrough indicating efficient decoding of Sec codon. Results indicated a temporal regulation of the selenoproteome with a general increase in their expression in inflamed cells in a Se-dependent manner. Selenow, Gpx1, Msrb1, and Selenom were highly upregulated upon stimulation with LPS when compared to other selenoproteins. Interestingly, Selenow appeared to be one amongst the highly regulated selenoproteins in macrophages that was previously thought to be mainly restricted to myocytes. Collectively, TMT-labeling method of non-Sec peptides offers a reliable method to quantitate and study temporal regulation of selenoproteins; however, further optimization to include Sec-peptides could make this strategy more robust and sensitive compared to other semi-quantitative or qualitative methods. Graphical Abstract.
硒（Se）作为第 21 种氨基酸——硒代半胱氨酸（Sec）掺入蛋白质中，起到细胞内氧化还原门控的作用。用外源性硒（亚硒酸钠）补充巨噬细胞会下调炎症和细胞内氧化应激，因为它能在受到细菌内毒素脂多糖（LPS）刺激时有效地恢复氧化还原平衡。在这里，我们使用标准的串联质量标签（TMT）标记质谱基于蛋白质组学的工作流程来定量和检查这种炎症细胞中硒蛋白的时间调节。使用缺乏硒的小鼠原代骨髓来源的巨噬细胞（BMDM），在 LPS 存在或不存在亚硒酸钠处理的情况下，在不同的时间段（0-20 小时）暴露于 LPS，使用等压标记和鸟枪法蛋白质组学工作流程分析硒蛋白组表达。为了克服 Sec 肽鉴定的挑战，我们使用 Sec 下游非 Sec 肽的鉴定作为核糖体通读的可靠证据，表明 Sec 密码子的有效解码。结果表明硒蛋白组具有时间调节性，其在炎症细胞中的表达普遍增加，这是一种依赖于硒的方式。与其他硒蛋白相比，在受到 LPS 刺激时，硒氧还酶、谷胱甘肽过氧化物酶 1、Msrb1 和 Selenom 的表达明显上调。有趣的是，与以前认为主要局限于肌细胞的观点相反，Selenow 似乎是巨噬细胞中高度调节的硒蛋白之一。总的来说，非 Sec 肽的 TMT 标记方法为定量和研究硒蛋白的时间调节提供了一种可靠的方法；然而，与其他半定量或定性方法相比，进一步优化包括 Sec 肽可以使这种策略更稳健和敏感。
