Segment Scan Mass Spectral Acquisition for Increasing the Metabolite Detectability in Chemical Isotope Labeling Liquid Chromatography–Mass Spectrometry Metabolome Analysis

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.

本研究报道了一种应用于化学同位素标记（Chemical Isotope Labeling, CIL）液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry, LC–MS）中的轨道阱质谱（Orbitrap MS）分段扫描方法，以提升代谢物检测效率。该方法将完整的质荷比（m/z）范围划分为多个区段，在液相色谱数据采集过程中对每个区段单独扫描，从而获得多张窄范围质谱图。对这些分段扫描得到的质谱图分别进行处理，以提取峰对信息：每一组峰对均来自经¹³C与¹²C试剂标记的混合样品中被差异标记的代谢物。将各峰对子列表进行合并，即可得到本次LC–MS分析的最终峰对列表。本研究考察了多项实验参数，包括自动增益控制（Automatic Gain Control, AGC）参数、质谱分辨率、区段m/z宽度、区段数量以及液相色谱运行的总数据采集时间，旨在确定分段扫描的最优参数设置，在保持与全扫描一致的分析时长的前提下，提升可检测到的代谢物数量。针对16分钟的CIL LC–MS分析流程，最优参数设置为区段宽度120 m/z、分辨率60k。以丹磺酰基（dansyl）标记的人尿液样品为例，本研究证实：相较于全扫描模式下可检测到的3765组峰对，该分段扫描方法可检测到5867组峰对（即代谢物，而非特征峰），提升幅度达56%。在这5867组峰对中，有5575组（占比95.0%）可被鉴定或通过质荷比匹配确认。由于分段扫描纳入了更多低丰度峰对，相对定量准确性略有下降：全扫描模式下有87%的峰对其峰比值落在预期值1.0的±25%范围内，而分段扫描模式下该比例为81%。分段扫描并未对峰比值的测量精度产生显著影响。此外，当采用多次重复进样时，在分辨率设置为120k、区段宽度为60 m/z的轨道阱质谱上，该方法可将全扫描模式下可检测的峰对数量从3843组提升至7273组，提升幅度达89%。综上，轨道阱质谱分段扫描方法可有效提升CIL LC–MS分析中共流出代谢物的检测能力，进而扩大代谢组学覆盖范围。