Deep Coverage of Global Protein Expression and Phosphorylation in Breast Tumor Cell Lines Using TMT 10-plex Isobaric Labeling.
Deep Coverage of Global Protein Expression and Phosphorylation in Breast Tumor Cell Lines Using TMT 10-plex Isobaric Labeling.
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DOI：
10.1021/acs.jproteome.6b00374
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发表时间：
2017-03-03
影响因子：
4.4
通讯作者：
Neubert TA
中科院分区：
文献类型：
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作者：
Huang FK;Zhang G;Lawlor K;Nazarian A;Philip J;Tempst P;Dephoure N;Neubert TA
用同量异位标签标记肽是定量自下而上蛋白质组学中的一种流行策略。在本研究中，我们使用 10 重串联质量标签试剂标记了 6 种乳腺肿瘤细胞裂解物（每个通道 1.34 毫克蛋白质），并在 Q Exactive HF 四极轨道阱质谱仪上分析了样品。我们总共鉴定了 8706 个蛋白质和 28186 个磷酸肽，其中包括所有通道共有的 7394 个蛋白质和 23739 个磷酸位点。大多数技术重复与 R2 ≥ 0.98 相关，表明标记后引入了最小的变异性。磷酸肽数据集的无监督分层聚类成功地将乳腺肿瘤样本分为 Her2（表皮生长因子受体 2）阳性组和 Her2 阴性组，而 mRNA 丰度则不然。 Her2阳性组的受体酪氨酸激酶、磷酸肌醇3激酶、蛋白激酶Cδ和Src同源2等的酪氨酸磷酸化水平显着高于Her2阴性组。尽管在基于 MS2 的实验中进行了比率压缩，但我们证明使用 MS2 方法计算的比率与使用基于 MS3 的定量（使用 Thermo Orbitrap Fusion 质谱仪）减少比率抑制获得的比率高度相关（R2 > 0.65）。鉴于全局和磷酸化蛋白质组的深度覆盖，我们的数据表明，使用 TMT 的基于 MS2 的定量可以成功用于大规模多重定量蛋白质组学。
Labeling peptides with isobaric tags is a popular strategy in quantitative bottom-up proteomics. In this study, we labeled six breast tumor cell lysates (1.34 mg proteins per channel) using 10-plex tandem mass tag reagents and analyzed the samples on a Q Exactive HF Quadrupole-Orbitrap mass spectrometer. We identified a total of 8706 proteins and 28186 phosphopeptides, including 7394 proteins and 23739 phosphosites common to all channels. The majority of technical replicates correlated with a R2 ≥ 0.98, indicating minimum variability was introduced after labeling. Unsupervised hierarchical clustering of phosphopeptide datasets successfully classified the breast tumor samples into Her2 (epidermal growth factor receptor 2) positive and Her2 negative groups, whereas mRNA abundance did not. The tyrosine phosphorylation levels of receptor tyrosine kinases, phosphoinositide-3-kinase, protein kinase C delta and Src homology 2, among others, were significantly higher in the Her2 positive than the Her2 negative group. Despite ratio compression in MS2-based experiments, we demonstrated the ratios calculated using an MS2 method are highly correlated (R2 > 0.65) with ratios obtained using MS3-based quantitation (using a Thermo Orbitrap Fusion mass spectrometer) with reduced ratio suppression. Given the deep coverage of global and phosphoproteomes, our data show that MS2-based quantitation using TMT can be successfully used for large-scale multiplexed quantitative proteomics.