Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK.
Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, UK.
Rapid Commun Mass Spectrom. 2022 Jul 15;36(13):e9308. doi: 10.1002/rcm.9308.
Analyte quantitation by mass spectrometry underpins a diverse range of scientific endeavors. The fast-growing field of mass spectrometer development has resulted in several targeted and untargeted acquisition modes suitable for these applications. By characterizing the acquisition methods available on an ion mobility (IM)-enabled orthogonal acceleration time-of-flight (oa-ToF) instrument, the optimum modes for analyte semi-quantitation can be deduced.
Serial dilutions of commercial metabolite, peptide, or cross-linked peptide analytes were prepared in matrices of human urine or Escherichia coli digest. Each analyte dilution was introduced into an IM separation-enabled oa-ToF mass spectrometer by reversed-phase liquid chromatography and electrospray ionization. Data were acquired for each sample in duplicate using nine different acquisition modes, including four IM-enabled acquisitions modes, available on the mass spectrometer.
Five (metabolite) or seven (peptide/cross-linked peptide) point calibration curves were prepared for analytes across each of the acquisition modes. A nonlinear response was observed at high concentrations for some modes, attributed to saturation effects. Two correction methods, one MS1 isotope-correction and one MS2 ion intensity-correction, were applied to address this observation, resulting in an up to twofold increase in dynamic range. By averaging the semi-quantitative results across analyte classes, two parameters, linear dynamic range (LDR) and lower limit of quantification (LLOQ), were determined to evaluate each mode.
A comparison of the acquisition modes revealed that data-independent acquisition and parallel reaction monitoring methods are most robust for semi-quantitation when considering achievable LDR and LLOQ. IM-enabled modes exhibited sensitivity increases, but a simultaneous reduction in dynamic range required correction methods to recover. These findings will assist users in identifying the optimum acquisition mode for their analyte quantitation needs, supporting a diverse range of applications and providing guidance for future acquisition mode developments.
质谱分析物定量是多种科学研究的基础。快速发展的质谱仪开发领域产生了几种适用于这些应用的靶向和非靶向采集模式。通过对具有离子淌度（IM）功能的正交加速飞行时间（oa-ToF）仪器上可用的采集方法进行特征描述，可以推导出用于分析物半定量的最佳模式。
在人类尿液或大肠杆菌消化物的基质中制备商业代谢物、肽或交联肽分析物的系列稀释液。将每种分析物稀释液通过反相液相色谱和电喷雾电离引入到具有 IM 分离功能的 oa-ToF 质谱仪中。使用质谱仪上提供的 9 种不同采集模式中的每一种对每个样品进行重复采集。
为每种采集模式下的分析物准备了五个（代谢物）或七个（肽/交联肽）点校准曲线。对于某些模式，在高浓度下观察到非线性响应，归因于饱和效应。应用了两种校正方法，一种是 MS1 同位素校正，另一种是 MS2 离子强度校正，以解决这一观察结果，导致动态范围增加了一倍。通过对分析物类别进行半定量结果的平均处理，确定了两个参数，线性动态范围（LDR）和定量下限（LLOQ），以评估每种模式。
对采集模式的比较表明，在考虑可实现的 LDR 和 LLOQ 时，数据非依赖性采集和并行反应监测方法对于半定量最稳健。具有 IM 功能的模式表现出灵敏度的提高，但同时动态范围的缩小需要校正方法来恢复。这些发现将有助于用户确定最适合其分析物定量需求的采集模式，支持多种应用，并为未来的采集模式开发提供指导。
