PDF(Pubmed)
Abstract：
For targeted protein panels, the ability to specifically assay post-translational modifications (PTMs) in a quantitative, sensitive, and straightforward manner would substantially advance biological and pharmacological studies. The present study highlights the effectiveness of the Affi-BAMS™ epitope-directed affinity bead capture/MALDI MS platform for quantitatively defining complex PTM marks of H3 and H4 histones. Using H3 and H4 histone peptides and isotopically labelled derivatives, this affinity bead and MALDI MS platform achieves a range of >3 orders of magnitude with a technical precision CV of <5%. Using nuclear cellular lysates, Affi-BAMS PTM-peptide capture resolves heterogeneous histone N-terminal PTMs with as little as 100 µg of starting material. In an HDAC inhibitor and MCF7 cell line model, the ability to monitor dynamic histone H3 acetylation and methylation events is further demonstrated (including SILAC quantification). Affi-BAMS (and its capacity for the multiplexing of samples and target PTM-proteins) thus provides a uniquely efficient and effective approach for analyzing dynamic epigenetic histone marks, which is critical for the regulation of chromatin structure and gene expression.
摘要：
对于靶向蛋白质面板，定量特异性测定翻译后修饰（PTM）的能力，敏感,和直接的方式将大大推进生物学和药理学研究。本研究强调了Affi-BAMS™表位定向亲和珠捕获/MALDIMS平台用于定量定义H3和H4组蛋白的复杂PTM标记的有效性。使用H3和H4组蛋白肽和同位素标记的衍生物，该亲和珠和MALDIMS平台实现了>3个数量级的范围,技术精度CV<5%。使用核细胞裂解物，Affi-BAMSPTM-肽捕获解决了异质组蛋白N端PTM，只有100µg的起始材料。在HDAC抑制剂和MCF7细胞系模型中，进一步证明了监测动态组蛋白H3乙酰化和甲基化事件的能力(包括SILAC定量).因此，Affi-BAMS（及其样品和靶PTM蛋白的多路复用能力）为分析动态表观遗传组蛋白标记提供了独特的有效方法，这对于调节染色质结构和基因表达至关重要。
