定义 TNF-α 和 IL-1β 诱导的新生蛋白：结合生物正交非规范氨基酸标记和蛋白质组学。
Defining TNF-α and IL-1β induced nascent proteins: combining bio-orthogonal non-canonical amino acid tagging and proteomics.
机构信息
Manitoba Centre for Proteomics and Systems Biology, Department of Internal Medicine, Winnipeg, MB, Canada.
出版信息
J Immunol Methods. 2012 Aug 31;382(1-2):189-95. doi: 10.1016/j.jim.2012.06.003. Epub 2012 Jun 12.
An impediment in the development of new therapeutic strategies for chronic inflammatory diseases is the limited understanding of underlying molecular mechanisms. The objective of this study was to identify newly synthesized (nascent) proteins induced by critical inflammatory cytokines TNF-α and IL-1β in human monocytic THP-1 cells. We optimized methods to combine two different approaches, bio-orthogonal non-canonical amino acid tagging (BONCAT) along with proteomics using isobaric tags (iTRAQ). BONCAT employed the incorporation of l-azidohomoalanine (AHA), an analog of methionine, into TNF-α or IL-1β induced nascent proteins. The AHA-containing nascent proteins were tagged with alkyne-biotin to allow enrichment using avidin affinity purification. The differential expressions of the enriched proteins were further determined using iTRAQ reagents and mass spectrometry (MS). The combination of BONCAT and proteomics represents a unique approach that has uncovered the nascent proteome induced by inflammatory cytokines TNF-α and IL-1β.
在开发慢性炎症性疾病新治疗策略方面的一个障碍是对潜在分子机制的理解有限。本研究的目的是鉴定由关键炎症细胞因子 TNF-α 和 IL-1β 在人单核细胞 THP-1 细胞中诱导的新合成（新生）蛋白质。我们优化了方法，将两种不同的方法结合起来，即生物正交非典型氨基酸标记（BONCAT）和使用等重同位素标记相对和绝对定量技术（iTRAQ）的蛋白质组学。BONCAT 采用将 l-叠氮高丙氨酸（AHA），一种蛋氨酸类似物，掺入到 TNF-α 或 IL-1β 诱导的新生蛋白质中。含有 AHA 的新生蛋白质与炔基生物素标记，以便使用亲和素亲和纯化进行富集。用 iTRAQ 试剂和质谱（MS）进一步确定富集蛋白质的差异表达。BONCAT 和蛋白质组学的结合代表了一种独特的方法，它揭示了炎症细胞因子 TNF-α 和 IL-1β 诱导的新生蛋白质组。
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