The Novo Nordisk Foundation Center for Protein Research , University of Copenhagen , 2200 Copenhagen , Denmark.
Thermo Fisher Scientific , 28199 Bremen , Germany.
J Proteome Res. 2018 Nov 2;17(11):4008-4016. doi: 10.1021/acs.jproteome.8b00381. Epub 2018 Sep 28.
A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT), which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here, we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments, relying on isobaric TMT reporter ion quantification.
一种常用的肽定量方法依赖于同位标记，如串联质量标签（TMT），这使得能够进行多重蛋白质组分析。通过串联质谱仪中的碎片产生报告离子来实现定量。然而，随着多重化程度的增加，报告离子之间较小的质量差异会增加质量分辨率的要求。这与轨道阱仪器降低质量分辨率所带来的更快的肽测序能力形成对比。因此，在最大限度地提高蛋白质组深度时，确定高度多重化定量的质量分辨率限制非常重要。在这里，我们使用超高场轨道阱质谱仪定义了分辨率为 0.0063 Da 的 TMT 报告离子的下限。我们发现，最佳方法取决于紧密间隔的报告离子之间的相对比例，并且 64 ms 的瞬态采集时间为分离 TMT 报告离子提供了足够的分辨率，对于绝对比例变化高达 16 倍的报告离子也能够有效分离。此外，使用相位约束谱解卷积处理的 32 ms 瞬态提供了 >50%的更多鉴定，且具有 >99%的定量准确率，但定量精度和准确性略有下降。这些发现应该指导未来蛋白质组学实验中根据依赖于同位标记的 TMT 报告离子定量来选择何种轨道阱分辨率设置。
