Tailoring to Search Engines: Bottom-Up Proteomics with Collision Energies Optimized for Identification Confidence.
Tailoring to Search Engines: Bottom-Up Proteomics with Collision Energies Optimized for Identification Confidence.
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DOI：
10.1021/acs.jproteome.0c00518
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发表时间：
2021-01-01
影响因子：
4.4
通讯作者：
Drahos L
中科院分区：
文献类型：
--
作者：
Révész Á;Milley MG;Nagy K;Szabó D;Kalló G;Csősz É;Vékey K;Drahos L
自下而上的蛋白质组学依赖于从串联质谱中识别肽，通常通过与序列数据库匹配。肽谱匹配的置信度可以由数据库搜索引擎给出的得分值来表征，并且它取决于谱的信息内容和质量。后者受实验参数的影响，其中碰撞能是最重要的碰撞诱导解离的情况下。我们研究了Byonic和Andromeda（MaxQuant）引擎的识别分数如何随QTof仪器上HeLa胰蛋白酶消化的一千多个单独肽的碰撞能量而变化。因此，我们扩展了我们早期对吉祥物分数的研究，并证实了其对这种能量依赖的潜在双峰性质的发现。作为m/z函数的最佳能量显示出三种发动机的可比线性趋势。肽水平的结果的基础上，我们设计了一个或两个液相色谱-串联质谱（LC-MS/MS）运行和各种碰撞能量设置的方法，并评估其实际性能的肽和蛋白质鉴定的HeLa标准样品。与出厂默认设置相比，在各种测量中获得了10-40%的增益，例如鉴定的蛋白质数量或序列覆盖率。三种发动机的最佳性能方法不同，这表明实验参数应根据发动机的选择进行微调。我们还推荐了一种简单的方法，并提供了参考数据，以便于将优化的方法转移到与蛋白质组学相关的其他质谱仪上。我们证明了这种方法的实用性上的Orbitrap仪器。可通过MassIVE存储库（MSV 000086379）访问数据集。
Bottom-up proteomics relies on identification of peptides from tandem mass spectra, usually via matching against sequence databases. Confidence in a peptide–spectrum match can be characterized by a score value given by the database search engines, and it depends on the information content and the quality of the spectrum. The latter are influenced by experimental parameters, of which the collision energy is the most important one in the case of collision-induced dissociation. We examined how the identification score of the Byonic and Andromeda (MaxQuant) engines varies with collision energy for more than a thousand individual peptides from a HeLa tryptic digest on a QTof instrument. We thereby extended our earlier study on Mascot scores and corroborated its findings on the potential bimodal nature of this energy dependence. Optimal energies as a function of m/z show comparable linear trends for the three engines. On the basis of peptide-level results, we designed methods with one or two liquid chromatography–tandem mass spectrometry (LC-MS/MS) runs and various collision energy settings and assessed their practical performance in peptide and protein identification from the HeLa standard sample. A 10–40% gain in various measures, such as the number of identified proteins or sequence coverage, was obtained over the factory default settings. Best performing methods differ for the three engines, suggesting that the experimental parameters should be fine-tuned to the choice of the engine. We also recommend a simple approach and provide reference data to ease the transfer of the optimized methods to other mass spectrometers relevant for proteomics. We demonstrate the utility of this approach on an Orbitrap instrument. Data sets can be accessed via the MassIVE repository (MSV000086379).
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优化科学工作流程，改进多肽和蛋白质鉴定。
影响因子：
3
作者：
Holl S;Mohammed Y;Zimmermann O;Palmblad M
通讯作者：
Palmblad M
DOI：
10.1074/mcp.o113.034181
发表时间：
2013-12
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Olsen JV;Mann M
通讯作者：
Mann M
影响因子：
4.4
作者：
Cox, Juergen;Neuhauser, Nadin;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
4.4
作者：
Holstein (Sherwood), Carly A.;Gafken, Philip R.;Martin, Daniel B.
通讯作者：
Martin, Daniel B.
DOI：
10.1016/j.jasms.2008.11.005
发表时间：
2009-03-01
影响因子：
3.2
作者：
Neta, Pedatsur;Simon-Manso, Yamil;Stein, Stephen E.
通讯作者：
Stein, Stephen E.