Department of Chemistry, University of Natural Resources and Life Sciences - BOKU Vienna, Muthgasse 18, 1190, Vienna, Austria.
Agilent Technologies, 5301 Stevens Creek Blvd, Santa Clara, CA, 95051, USA.
Rapid Commun Mass Spectrom. 2019 Jul;33 Suppl 2(Suppl Suppl 2):66-74. doi: 10.1002/rcm.8420. Epub 2019 May 2.
The wide chemical diversity and complex matrices inherent to metabolomics still pose a challenge to current analytical approaches for metabolite screening. Although dedicated front-end separation techniques combined with high-resolution mass spectrometry set the benchmark from an analytical point of view, the increasing number of samples and sample complexity demand for a compromise in terms of selectivity, sensitivity and high-throughput analyses.
Prior to low-field drift tube ion mobility (IM) separation and quadrupole time-of-flight mass spectrometry (QTOFMS) detection, rapid ultrahigh-performance liquid chromatography separation was used for analysis of different concentration levels of dansylated metabolites present in a yeast cell extract. For identity confirmation of metabolites at the MS2 level, an alternating frame approach was chosen and two different strategies were tested: a data-independent all-ions acquisition and a quadrupole broad band isolation (Q-BBI) directed by IM drift separation.
For Q-BBI analysis, the broad mass range isolation was successfully optimized in accordance with the distinctive drift time to m/z correlation of the dansyl derivatives. To guarantee comprehensive sampling, a broad mass isolation window of 70 Da was employed. Fragmentation was performed via collision-induced dissociation, applying a collision energy ramp optimized for the dansyl derivatives. Both approaches were studied in terms of linear dynamic range and repeatability employing ethanolic extracts of Pichia pastoris spiked with 1 μM metabolite mixture. Example data obtained for histidine and glycine showed that drift time precision (<0.01 to 0.3% RSD, n = 5) compared very well with the data reported in an earlier IM-TOFMS-based study.
Chimeric mass spectra, inherent to data-independent analysis approaches, are reduced when using a drift time directed Q-BBI approach. Additionally, an improved linear dynamic working range was observed, representing, together with a rapid front-end separation, a powerful approach for metabolite screening.
代谢组学中广泛的化学多样性和复杂的基质仍然对当前的代谢物筛选分析方法构成挑战。虽然专用前端分离技术与高分辨率质谱相结合从分析角度来看设定了基准，但越来越多的样品和样品复杂性要求在选择性、灵敏度和高通量分析方面做出妥协。
在低场漂移管离子迁移（IM）分离和四极杆飞行时间质谱（QTOFMS）检测之前，快速超高液相色谱分离用于分析酵母细胞提取物中存在的不同浓度水平的丹酰化代谢物。为了在 MS2 水平上确认代谢物的身份，选择了交替帧方法，并测试了两种不同的策略：一种是数据独立的全离子采集，另一种是由 IM 漂移分离指导的四极宽带隔离（Q-BBI）。
对于 Q-BBI 分析，根据丹酰衍生物独特的漂移时间与 m/z 相关性，成功优化了宽质量范围的隔离。为了保证全面采样，采用了 70 Da 的宽质量隔离窗口。通过碰撞诱导解离进行碎片化，应用针对丹酰衍生物优化的碰撞能斜坡。使用毕赤酵母乙醇提取物中添加 1 μM 代谢物混合物，研究了两种方法的线性动态范围和重复性。对于组氨酸和甘氨酸获得的示例数据表明，漂移时间精度（<0.01 至 0.3%RSD，n=5）与基于 IM-TOFMS 的早期研究中报告的数据非常吻合。
当使用基于漂移时间的 Q-BBI 方法时，固有数据独立分析方法的嵌合质谱会减少。此外，观察到改进的线性动态工作范围，与快速前端分离一起，代表了代谢物筛选的强大方法。
