We report a segmented spectrum scan method using Orbitrap MS in chemical isotope labeling (CIL) liquid chromatography–mass spectrometry (LC–MS) for improving the metabolite detection efficiency. In this method, the full m/z range is divided into multiple segments with the scanning of each segment to produce multiple narrow-range spectra during the LC data acquisition. These segmented spectra are separately processed to extract the peak pair information with each peak pair arising from a differentially labeled metabolite in the analysis of a mixture of ¹³C and ¹²C reagent-labeled samples. The sublists of peak pairs are merged to form the final peak pair list from the LC–MS run. Various experimental conditions, including automatic gain control (AGC) values, mass resolutions, segment m/z widths, number of segments, and total data acquisition time in the LC run, were examined to arrive at an optimal setting in the segment scan for increasing the number of detectable metabolites while maintaining the same analysis time as in the full scan. The optimal method used a segment width of 120 m/z with 60k resolution for a 16 min CIL LC–MS run. Using dansyl-labeled human urine samples as an example, we demonstrated that this method could detect 5867 peak pairs or metabolites (not features), compared to 3765 peak pairs detectable in a full scan, representing a 56% gain. Out of 5867 peak pairs, 5575 (95.0%) could be identified or mass-matched. The relative quantification accuracy was slightly reduced (81% peak pairs were within ±25% of the expected peak ratio of 1.0 in full, compared to 87% in the full scan) due to the inclusion of more low-abundance peak pairs in the segment scan. The peak ratio measurement precision was not significantly affected by the segment scan. We also showed the increase of the peak pair number detectable from 3843 in the full scan to 7273 (89% gain) using the Orbitrap operated at 120k resolution with a 60 m/z segment width when multiple repeat sample injections were used. Thus, segment scan Orbitrap MS is an enabling method for detecting coeluting metabolites in CIL LC–MS for increasing the metabolomic coverage.

中文翻译：

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用于提高化学同位素标记液相色谱-质谱代谢组分析中代谢物检测能力的分段扫描质谱采集

我们报告了一种在化学同位素标记 (CIL) 液相色谱-质谱 (LC-MS) 中使用 Orbitrap MS 的分段光谱扫描方法，以提高代谢物检测效率。在这种方法中，整个m / z范围被分成多个段，每个段的扫描在 LC 数据采集过程中产生多个窄范围光谱。^{这些分段的光谱被单独处理以提取峰对信息，每个峰对在13} C 和¹²的混合物的分析中产生于差异标记的代谢物C 试剂标记的样品。合并峰对的子列表以形成来自 LC-MS 运行的最终峰对列表。检查各种实验条件，包括自动增益控制 (AGC) 值、质量分辨率、分段m / z宽度、分段数和 LC 运行中的总数据采集时间，以在分段扫描中达到最佳设置以增加可检测代谢物的数量，同时保持与全扫描相同的分析时间。最佳方法使用 120 m / z的线段宽度以 60k 分辨率进行 16 分钟的 CIL LC-MS 运行。以丹磺酰标记的人尿液样本为例，我们证明了该方法可以检测到 5867 个峰对或代谢物（不是特征），而在全扫描中可检测到 3765 个峰对，代表了 56% 的增益。在 5867 个峰对中，5575 个 (95.0%) 可以被识别或质量匹配。由于在片段中包含更多低丰度峰对，相对定量准确度略有降低（81% 峰对在完全预期峰比 1.0 的 ±25% 范围内，而全扫描中为 87%）扫描。峰比测量精度不受分段扫描的显着影响。我们还展示了使用 Orbitrap 在 120k 分辨率和 60使用多次重复进样时的m / z段宽度。因此，分段扫描 Orbitrap MS 是一种在 CIL LC-MS 中检测共洗脱代谢物以增加代谢组学覆盖率的有效方法。