Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3,Journal of Proteome Research

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Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2018-05-16 , DOI: 10.1021/acs.jproteome.8b00217
José Navarrete-Perea ₁ , Qing Yu ₁ , Steven P. Gygi ₁ , Joao A. Paulo ₁

Affiliation

Mass spectrometry (MS) coupled toisobaric labeling has developed rapidly into a powerful strategy for high-throughput protein quantification. Sample multiplexing and exceptional sensitivity allow for the quantification of tens of thousands of peptides and, by inference, thousands of proteins from multiple samples in a single MS experiment. Accurate quantification demands a consistent and robust sample-preparation strategy. Here, we present a detailed workflow for SPS-MS3-based quantitative abundance profiling of tandem mass tag (TMT)-labeled proteins and phosphopeptides that we have named the streamlined (SL)-TMT protocol. We describe a universally applicable strategy that requires minimal individual sample processing and permits the seamless addition of a phosphopeptide enrichment step (“mini-phos”) with little deviation from the deep proteome analysis. To showcase our workflow, we profile the proteome of wild-type Saccharomyces cerevisiae yeast grown with either glucose or pyruvate as the carbon source. Here, we have established a streamlined TMT protocol that enables deep proteome and medium-scale phosphoproteome analysis.

中文翻译：

简化的串联质量标签（SL-TMT）协议：使用串联质量标签同步前体选择-MS3进行定量（磷酸）蛋白质组分析的有效策略

质谱（MS）结合等压标记已迅速发展成为一种高通量蛋白质定量的强大策略。样品的多路复用和出色的灵敏度可在一次MS实验中定量分析成千上万的肽，并通过推理对多个样品中的成千上万的蛋白质进行定量。准确的定量需要一种一致且稳健的样品制备策略。在这里，我们为串联质量标签（TMT）标记的蛋白质和磷酸肽提供基于SPS-MS3的定量丰度分析的详细工作流程，我们将其命名为简化的（SL）-TMT协议。我们描述了一种普遍适用的策略，该策略要求最少的单个样本处理并允许无缝添加磷酸肽富集步骤（“ mini-phos”），而与深度蛋白质组分析的偏差很小。为了展示我们的工作流程，我们介绍了野生型蛋白质组以葡萄糖或丙酮酸为碳源生长的酿酒酵母酵母。在这里，我们已经建立了简化的TMT协议，可以进行深层蛋白质组和中等规模的磷酸化蛋白质组分析。

更新日期：2018-05-16

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