Thermal proteome profiling is a powerful energetic‐based chemical proteomics method to reveal the ligand‐protein interaction. However, the costly multiplexed isotopic labeling reagent, mainly Multiplexed isobaric tandem mass tag (TMT), and the long mass spectrometric time limits the wide application of this method. Here a simple and cost‐effective strategy by using dimethyl labeling technique instead of TMT labeling is reported to quantify proteins and by using the peptides derived from the same protein to determine significantly changed proteins in one LC‐MS run. This method is validated by identifying the known targets of methotrexate and geldanamycin. In addition, several potential off‐targets involved in detoxification of reactive oxygen species pathway are also discovered for geldanamycin. This method is further applied to map the interactome of adenosine triphosphate (ATP) in the 293T cell lysate by using ATP analogue, adenylyl imidodiphosphate (AMP‐PNP), as the ligand. As a result, a total of 123 AMP‐PNP‐sensitive proteins are found, of which 59 proteins are stabilized by AMP‐PNP. Approximately 53% and 20% of these stabilized candidate protein targets are known as ATP and RNA binding proteins. Overall, above results demonstrated that this approach could be a valuable platform for the unbiased target proteins identification with reduced reagent cost and mass spectrometric time.

中文翻译：

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用于筛选配体蛋白质靶标的简化热蛋白质组分析方法。


热蛋白质组分析是一种强大的基于能量的化学蛋白质组学方法，用于揭示配体-蛋白质相互作用。然而，多重同位素标记试剂（主要是多重同量异位串联质量标签（TMT））价格昂贵，且质谱分析时间长限制了该方法的广泛应用。据报道，一种简单且经济有效的策略，即使用二甲基标记技术而不是 TMT 标记来量化蛋白质，并使用源自同一蛋白质的肽来确定一次 LC-MS 运行中显着变化的蛋白质。该方法通过识别甲氨蝶呤和格尔德霉素的已知靶点进行验证。此外，格尔德霉素还发现了一些参与活性氧解毒途径的潜在脱靶。该方法进一步应用 ATP 类似物腺苷酰亚胺二磷酸 (AMP-PNP) 作为配体来绘制 293T 细胞裂解液中三磷酸腺苷 (ATP) 的相互作用组图。结果，总共发现了 123 个 AMP-PNP 敏感蛋白，其中 59 个蛋白被 AMP-PNP 稳定。这些稳定的候选蛋白靶点中大约 53% 和 20% 被称为 ATP 和 RNA 结合蛋白。总体而言，上述结果表明，该方法可能是一个有价值的平台，可以减少试剂成本和质谱时间，用于无偏见的目标蛋白鉴定。