文章
Paper背景
蛋白质组学已逐渐成为解决生物学问题强有力的工具,然而在很多技术上,蛋白质组学依旧落后于基因组学,如分析速度、组学深度及定量的准确性。
在相对定量蛋白质组学中,同位素标记试剂TMT允许对多个样本同时进行质谱分析,起初TMT最多只能用于6个样本(6-plex)的分析,随后,通过在C13上引入N15、调整5个重标的原子的位置,进一步增加了通道数目,TMT标记试剂可达11plex。但如果想要继续增加通道,则需要引入更多的重标及新的报告离子。此研究报告了新一代的TMT pro试剂,该试剂由9个重标原子组成,理论上可以组成18种不同的标签,文章主要报道了TMT pro 16-plex的性能。
Fig 1. TMT和TMT pro的组成
结果
- 首先利用单个细胞系对TMT0 和TMT pro0进行了简单的比较:
- 标记效率:TMT and TMTpro tags (that is, TMT0 and TMTpro0);
- 缺失值:Partially labeled (TMT0: 1.3%; TMTpro0: 1.9%) or unlabeled (TMT0: 3.2%; TMTpro0: 0.4%);
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从肽段鉴定数目、碰撞能量NCE、S/N、SEQUEST scores、试剂稳定性等方面对两种试剂进行了系统比较,发现虽然TMT pro鉴定到的肽段少一些,但其他性能与TMT相当,甚至优于TMT;
TMT0和TMT pro0对比
- 利用两种细胞系两种处理组和两种质谱扫描技术比较两种试剂定量的精确性
- 实验设计来自AC上 MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes.
实验设计及部分结果 -
使用SPS–MS3定量技术: 平均CV均< 7%;对三组实验进行聚类分析,发现每种细胞系很好地聚在一起(hierarchical clustering analysis), 而且每个实验组中两种细胞系的比值相关系数都很好(Pearson correlation r=0.89, r=0.91),从11plex到16plex比值压缩效应并不明显;
使用a hrMS2 method was used on a Q-Exactive HF-X instrument: 相似的实验结果,Pearson correlation r=0.8, 0.78, 0.88, 0.85;
相似的实验结果
- 利用real-time search (RTS)技术,实现TMT pro 16plex在单次、复杂实验设计中的应用
- 实验设计及结果:应用RTS的优点是在不损失定量的情况下,减少了50%的分析时间;the streamlined–TMT-based protocol;8个细胞系2个处理条件3个生物学重复2个组学分析(蛋白质组和磷酸化蛋白质组);蛋白水平的质谱分析分三部分,预分了24份只分析了其中12份样本、24份全部分析、来自replicate 1的相同的12份样本分三种质谱分析;磷酸化蛋白质组分析,没有预分,利用iMAC富集;
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分析12份样本
图d,e:Unsupervised hierarchical clustering analysis (HCA) and principal component analysis (PCA) illustrated that samples were quantified without measurable bias or batch effect.
分析12份样本的结果 -
分析24份样本
Supplementary Figure 8c. protein expression was reproducible in the replicates but varied widely across cell lines,a two-way ANOVA revealed 8,484 significant cell type-specific protein expression differences (n=3 cell culture replicates, BH-adjusted p-value ≤ 1e-5, partial η2 ≥ 0.7).
d, A two-way ANOVA revealed that 2,027 proteins were significantly affected by both cell type and Torin1 (n=3 cell culture replicates, BH-adjusted p-value ≤ 1e-5, partial η2 ≥ 0.7).
e, A two- way ANOVA revealed 1,916 Torin1-specific protein expression differences (n=3 cell culture replicates, BH-adjusted p-value ≤ 1e-5, partial η2 ≥ 0.7). GO analysis showed down-regulated proteins were enriched in ribosome, translation and cell cycle, etc. Up-regulated proteins were enriched in lysosome, catabolic process and extracellular vesicle, etc. (Bonferroni-adjusted p-value ≤ 0.01, all quantified proteins were used as background).
分析全部样本的结果 -
蛋白质组三部分质谱技术相关性分析
Additionally, the 12 fractions from replicate 1 were reanalyzed using hrMS2 on a Q-Exactive HF-X instrument and SPS–MS3 on an Orbitrap Lumos instrument;
TMTpro reagents performed well with both methods, and presented linearly correlated quantitation between hrMS2, SPS–MS3 and RTS–SPS–MS3 methods;
Similar to classic TMT reagents, TMTpro reagents showed ratio compression with hrMS2;
结果
线性相关性和压缩效应
- 还有一个Thermal proteome profiling的实验例子,不再介绍....(没力气截图了😂)
统计学分析
质谱数据分析生物信息学分析
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