Metabolic labeling of plant cell cultures with K(15)NO3 as a tool for quantitative analysis of proteins and metabolites.
Metabolic labeling of plant cell cultures with K(15)NO3 as a tool for quantitative analysis of proteins and metabolites.
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用K（15）NO3作为蛋白质和代谢产物定量分析的工具，对植物细胞培养的代谢标记。
DOI：
10.1186/1746-4811-2-14
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发表时间：
2006-09-04
期刊：
影响因子：
5.1
通讯作者：
Schulze WX
中科院分区：
文献类型：
--
作者：
Engelsberger WR;Erban A;Kopka J;Schulze WX
在解决蛋白质列表之外的生物学问题的实验中，不同生物样品之间稳健的定量比较策略非常重要。在这里，我们建议使用 15N-KNO3 作为拟南芥细胞培养物中唯一的氮源，以实现代谢完全标记的细胞群。来自此类代谢标记培养物的蛋白质可通过特征质量转移与未标记的蛋白质群体区分开来，该特征质量转移取决于所分析的胰蛋白酶肽的氨基酸组成。此外，代谢标记的细胞提取物还适用于含氮细胞代谢补体的比较定量分析。将未标记细胞和标准化 15N 标记细胞的蛋白质提取物合并为一份样品，进行联合分析处理。其优点是 (i) 减少实验变异性和 (ii) 在单个提取的肽和代谢物光谱水平上进行直接相对定量。分析实验的相对定量的简易性和准确性都得到了显着提高。代谢标记策略已通过来自相同细胞培养物的蛋白质提取物和代谢物提取物的混合物得到验证，其中标记与未标记提取物的比例已知（1:1、1:4 和 4:1）。我们的结论是，饱和代谢 15N 标记提供了一种稳健且经济实惠的综合策略，可以回答定量蛋白质组学和氮聚焦代谢组学中的问题。
Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of 15N-KNO3 as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement. Protein extracts from unlabeled and from standardized 15N-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic 15N-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen focused metabolomics.
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影响因子：
3.4
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