Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples.

中文翻译：

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用于生物学和转化研究的快速和大规模泛素化分析。

蛋白质泛素化涉及大量的细胞过程。尽管针对遍在蛋白残基的抗体（K-G-GG）已经提高了使用质谱监测泛素化的能力，但是使用亚毫克量的样品进行组织和原代细胞中泛素化高度复用测量的方法仍然是一个挑战。在这里，我们提出了一种称为UbiFast的高度灵敏，快速和多路复用的方案，用于从大约500μg肽中每个样品中约10,000个泛素化位点定量，每个样品来自TMT10plex中约200个细胞或组织。5小时 高场非对称波形离子迁移谱（FAIMS）用于提高翻译后修饰分析的定量准确性。我们使用该方法重新发现靶向药物来那度胺的E3连接酶的底物，并确定在基础和管腔人类乳腺癌模型中被泛素化调节的蛋白质。UbiFast方法的灵敏度和速度使其适用于对原始组织样本进行大规模研究。