Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level.
Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level.
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超分辨率质谱可在 MS2 水平上实现快速、准确和高度多重的蛋白质组学。
DOI：
10.1021/acs.analchem.2c04742
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发表时间：
2023
影响因子：
7.4
通讯作者：
Tsybin,YuryO
中科院分区：
文献类型：
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作者：
Kozhinov,AntonN;Johnson,Alex;Nagornov,KonstantinO;Stadlmeier,Michael;Martin,WarhamLance;Dayon,Loïc;Corthésy,John;Wühr,Martin;Tsybin,YuryO
在基于串联质谱 (MS2) 的多重定量蛋白质组学中，开发了补体报告离子方法（TMTc 和 TMTproC），以消除传统 MS2 水平方法的比率压缩问题。解析所有高 m/z 补体报告离子（~6.32 mDa 间距）需要高于 OrbitrapTM 仪器性能水平的质量分辨率和扫描速度。因此，使用 TMT/TMTpro 试剂进行补体报告离子定量目前仅限于 11 个通道中的 5 个 (TMT) 或 18 个通道中的 9 个 (TMTpro)（~1 Da 间隔）。我们首先证明 FusionTMLumosTMOrbitrap 可以通过扩展 3 s 瞬态的标准采集模式解析 6.32 mDa 间距的补体报告离子。然后，我们使用最小二乘拟合 (LSF) 方法实施超分辨率质谱方法来处理 Orbitrap 瞬态，以实现鸟枪式蛋白质组学兼容的扫描速率。 LSF 性能可解析标准质量范围内所有 TMTproC 通道的 6.32 mDa 双峰，瞬态短至 ∼108 ms（Orbitrap 分辨率设置为 50,000 atm/z200）。然而，我们观察到与 1 Da 间距和 108 ms 瞬变相比，测量精度略有下降。对于 256 ms 瞬态（分辨率为 120,000 atm/z200），变异系数基本上与 1 Da 样本无法区分。因此，我们证明了在 MS2 水平上高度多重、准确和精确的鸟枪法蛋白质组学的可行性。
In tandem mass spectrometry (MS2)-based multiplexed quantitative proteomics, the complement reporter ion approaches (TMTc and TMTproC) were developed to eliminate the ratio-compression problem of conventional MS2-level approaches. Resolving all highm/zcomplement reporter ions (∼6.32 mDa-spaced) requires mass resolution and scan speeds above the performance levels of OrbitrapTMinstruments. Therefore, complement reporter ion quantification with TMT/TMTpro reagents is currently limited to 5 out of 11 (TMT) or 9 out of 18 (TMTpro) channels (∼1 Da spaced). We first demonstrate that a FusionTMLumosTMOrbitrap can resolve 6.32 mDa-spaced complement reporter ions with standard acquisition modes extended with 3 s transients. We then implemented a super-resolution mass spectrometry approach using the least-squares fitting (LSF) method for processing Orbitrap transients to achieve shotgun proteomics-compatible scan rates. The LSF performance resolves the 6.32 mDa doublets for all TMTproC channels in the standard mass range with transients as short as ∼108 ms (Orbitrap resolution setting of 50,000 atm/z200). However, we observe a slight decrease in measurement precision compared to 1 Da spacing with the 108 ms transients. With 256 ms transients (resolution of 120,000 atm/z200), coefficients of variation are essentially indistinguishable from 1 Da samples. We thus demonstrate the feasibility of highly multiplexed, accurate, and precise shotgun proteomics at the MS2 level.