A Multipathway Phosphopeptide Standard for Rapid Phosphoproteomics Assay Development.
A Multipathway Phosphopeptide Standard for Rapid Phosphoproteomics Assay Development.
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DOI：
10.1016/j.mcpro.2023.100639
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发表时间：
2023-10
影响因子：
7
通讯作者：
Patel, Bhavin
中科院分区：
文献类型：
--
作者：
Searle, Brian C.;Chien, Allis;Koller, Antonius;Hawke, David;Herren, Anthony W.;Kim, Jenny Kim;Lee, Kimberly A.;Leib, Ryan D.;Nelson, Alissa J.;Patel, Purvi;Ren, Jian Min;Stemmer, Paul M.;Zhu, Yiying;Neely, Benjamin A.;Patel, Bhavin
最近方法学上的进步使磷酸肽分析成为许多蛋白质组学研究人员面临的难题。现在有各种各样强大和可访问的浓缩策略来生成磷酸蛋白质组，而用于定量和位点定位的免费或廉价的软件工具极大地简化了磷蛋白质组分析工作流程。作为生物分子资源设施协会下属的一个研究小组，蛋白质组学标准研究组致力于开发一种基于重标记磷酸肽混合物的多途径磷酸肽标准，旨在使研究人员能够快速开发分析方法。这一混合物包含131个经过质谱学审查的磷酸肽，它们分别来自七个不同的信号网络：AMPK信号、死亡和凋亡信号、ErbB信号、胰岛素/胰岛素样生长因子-1信号、mTOR信号、PI3K/AKT信号和应激(p38/SAPK/JNK)信号。在这里，我们描述了将这种混合物添加到由表皮生长因子和胰岛素样生长因子-1刺激的HeLa胰酶消化液中，以激活MAPK和PI3K/AKT/mTOR通路的特征。我们进一步展示了使用这种与数据无关的获取的磷酸多肽混合物在五个实验室中独立执行的HeLa的磷酸蛋白质组学分析的比较。尽管实验和仪器过程不同，我们发现实验室可以通过报告测量与标准的比率来产生可重现的、协调的数据集，而强度测量显示实验室之间的一致性较低，即使在归一化之后。我们的结果表明，广泛可用的、与生物相关的磷肽标准可以作为跨实验室和样品制备的定量“标尺”，使更大规模的实验设计能够超过单个实验室的执行能力。原始数据文件在海量数据集MSV000090564中公开可用。131个重标记的多途径磷酸多肽标准品。五个实验室通过报告数据与标准的比率产生了协调一致的结果。协调比率明显比强度测量更一致。这一标准充当了实验室间的定量“标尺”。这种材料作为高纯度的商业标准公开可用。蛋白质组学标准研究小组开发了一种重标记磷肽的多途径标准混合物。这种混合物包含131个经过审查的磷酸肽，涵盖了来自七个不同信号网络的具有生物学意义的亚磷酸盐。我们在五个实验室中使用独立于数据的获取对这种混合物进行了表征。尽管实验过程不同，但实验室通过报告测量结果与标准的比率，产生了可重现的、协调的数据集。我们的结果表明，广泛可用的、与生物相关的标准可以作为实验室之间的定量“标尺”，使实验设计能够比单个实验室所能执行的更大。
Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative “yardstick” across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564. A multipathway phosphopeptide standard of 131 heavy-labeled phosphopeptides. Five labs produced harmonized results by reporting data as ratios to the standard. Harmonized ratios were significantly more consistent than intensity measurements. This standard acts as a quantitative “yardstick” across laboratories. The material is publicly available as a high-purity commercial standard. The Proteomics Standards Research Group developed a multipathway standard mixture of heavy-labeled phosphopeptides. This mixture contains 131 vetted phosphopeptides that cover biologically interesting phosphosites from seven different signaling networks. We characterized this mixture in five labs using data-independent acquisition. Despite different experimental processes, labs produced reproducible, harmonized datasets by reporting measurements as ratios to the standard. Our results suggest that widely available, biologically-relevant standards act as quantitative “yardsticks” across laboratories, enabling experimental designs larger than a single laboratory can perform.
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影响因子：
4.4
作者：
Hoopmann MR;Kusebauch U;Palmblad M;Bandeira N;Shteynberg DD;He L;Xia B;Stoychev SH;Omenn GS;Weintraub ST;Moritz RL
通讯作者：
Moritz RL
影响因子：
48
作者：
Gassaway, Brandon M.;Li, Jiaming;Rad, Ramin;Mintseris, Julian;Mohler, Kyle;Levy, Tyler;Aguiar, Mike;Beausoleil, Sean A.;Paulo, Joao A.;Rinehart, Jesse;Huttlin, Edward L.;Gygi, Steven P.
通讯作者：
Gygi, Steven P.
影响因子：
7
作者：
Huang, Ting;Bruderer, Roland;Reiter, Lukas
通讯作者：
Reiter, Lukas
使用 iRT（一种标准化保留时间）可以更有针对性地测量肽。
影响因子：
3.4
作者：
Escher, Claudia;Reiter, Lukas;MacLean, Brendan;Ossola, Reto;Herzog, Franz;Chilton, John;MacCoss, Michael J.;Rinner, Oliver
通讯作者：
Rinner, Oliver
影响因子：
46.9
作者：
Barber KW;Muir P;Szeligowski RV;Rogulina S;Gerstein M;Sampson JR;Isaacs FJ;Rinehart J
通讯作者：
Rinehart J