Azide-containing amino acid modulation of cellular transcriptome and proteome composition – implications on click chemistry based secretome analysis

The analysis of the secretome provides important information on cellular communication and on the recruitment and behavior of cells in specific tissues. Especially in the context of tumors, knowledge on the composition of the secretome plays an important role for diagnosis and therapy. Mass spectrometry-based analysis of cell-conditioned media is widely used for the unbiased characterization of cancer secretomes in vitro. Azide-containing amino acid labeling in combination with click chemistry now facilitates this type of analysis without serum starvation. The modified amino acids, however, are less efficiently incorporated into newly synthesized proteins. Utilizing transcriptome and proteome analysis, we describe here in detail the effects of labeling cells with the methionine analog azidohomoalanin (AHA) on the modulation of gene expression and protein composition. We observe that 11% – 34% of the proteins detected in the secretome were affected by AHA labeling regarding their expression levels in the transcriptome and/or proteome. Performing gene ontology analyses, our results provide a detailed view on the AHA-based induction of cellular stress and apoptosis-related pathways and provide first insights on how this might affect the composition of the secretome on a global scale. Overall design: To describe and understand the effects of AHA on the behaviour of cells in detail, we performed comparative transcriptome analyses of seven different tumor cells grown 20 h in AHA- or methionine-containing media and evaluated the gene expression profiles.

分泌组（secretome）分析可提供有关细胞通讯、特定组织内细胞募集与行为模式的关键信息。尤其在肿瘤研究领域，关于分泌组组成的认知对疾病诊断与治疗均具有重要价值。基于质谱法（mass spectrometry）的细胞条件培养基（cell-conditioned media）分析，是当前体外无偏表征癌症分泌组的常用实验方法。如今，叠氮修饰氨基酸标记技术结合点击化学（click chemistry），无需经过血清饥饿步骤即可开展此类分析。但这类修饰氨基酸在新合成蛋白质中的掺入效率相对偏低。
本研究结合转录组（transcriptome）与蛋白质组（proteome）分析，详细阐述了甲硫氨酸类似物叠氮高丙氨酸（AHA）对细胞基因表达调控与蛋白质组组成的影响。研究发现，分泌组中检测到的11%~34%蛋白质，其转录组和/或蛋白质组的表达水平会受到AHA标记的影响。通过基因本体论（gene ontology）分析，本研究结果详细揭示了AHA诱导的细胞应激与细胞凋亡相关通路，并首次从全局尺度阐明了该过程对分泌组组成的潜在调控作用。
实验整体设计：为全面解析AHA对细胞行为的具体影响，我们对7种分别在含AHA或甲硫氨酸的培养基中培养20小时的不同肿瘤细胞开展了对比转录组分析，并对其基因表达谱进行了系统评估。