prm-PASEF

抽象的

In comparison with standard selected and parallel reaction monitoring (SRM and PRM), the prm-PASEF increases the number of peptides that can be targeted in a single acquisition method, without compromising the selectivity or the sensitivity. Using a prototype acquisition software, we targeted 201 isotope-labeled synthetic peptides (AQUA peptides) spiked into a 100 ng human HeLa cell line digest. The prm-PASEF excelled with limits of quantification of 17,2 amol for some peptides utilizing an acquisition method that can monitor 216 precursors over a 30 min LC gradient. The new prm-PASEF method has a high reproducibility between injections and enables accurate quantification.

Authors

Antoine Lesur¹, Pierre-Olivier Schmit², Christopher Adams³，Gunnar Dittmar¹;
1 LIH, Strassen, Luxembourg; 2 Bruker France S.A. Wissembourg, France; 3 Bruker Daltonics Inc, San Jose, USA

Keywords:

4D-蛋白质组学，PRM-Pasef，Timstof Pro，Pasef，LFQ，绝对定量，靶向蛋白质组学

Targeted mass spectrometry is a powerful technique to support hypothesis-driven proteomics experiments, for instance, the verification of biomarker candidates in large sample cohorts. It alleviates the problem of missing values between samples but also increases the overall sensitivity compared to data-dependent (DDA) and data-independent (DIA) acquisition methods. Targeted proteomics uses synthetic peptides as an internal standard to normalize the MS signal and confirms the detection of the endogenous peptides with maximum confidence.

有针对性的蛋白质组学的主要限制是compromise that must be found between the number of targets measured in a single run, the duration of the liquid chromatography separation, and the overall sensitivity. This still holds true with the current generation of targeted acquisition methods including SRM and PRM. Therefore, it is only possible to achieve full data completeness for a large number of targeted peptides by either employing longer chromatographic separation or by compromising on the MS sensitivity and selectivity. Alternative approaches like DIA can partially address this issue and rely on a systematic co-isolation and co-*fragmentation of eluting peptides using broad m/z isolation windows. This approach measures most of the peptides, but with limited specificity and sensitivity (all fragments are mixed together) during the achievable MS cycles (the scanning speed of the analyzer has to be minimized) and is not easily combined with short chromatography gradients.

在本应用注释中，我们介绍了PRM-Pasef方法，这是Pasef采集策略的创新实现，该策略克服了与当前目标蛋白质组学技术相关的约束。

人类细胞系消化(100 ng /µL)飙升with 201 heavy aqua peptides and 15 light synthetic peptides as described in Figure 1. A dilution curve of nine concentration points ranging from 5.5 to 50,000 amol/µl was generated using 15 heavy AQUA peptides and their light counterpart. This set of peptides served as the internal standard. The other 186 AQUA peptides were spiked at the constant concentration of 2 fmol/µl in all samples.

将样品和对照三级注入技术，并在纳米HPLC（纳米ELUTE，BRUKER DALTONICS）上分离，使用250毫米拉的发射极柱（澳大利亚Ionopticks，澳大利亚），梯度梯度梯度为2-30％，范围为2-30％乙腈。在PRM-PASEF模式下运行的timstof Pro质谱仪（Bruker Daltonics）上分析肽。这项研究使用了单个PRM-PASEF方法：TIMS的积累时间固定为50 ms，而TIMS分离持续时间设置为100 ms。迁移率值的范围为0.65-1.3 1/k₀覆盖的m/z范围为100-1700m/z。每日天际线（20.1.1.83）处理数据。

评估定量性能，通过确定相应的浓度准确性和相对标准偏差来评估每个浓度水平下的15个重/光比。作为质量控制，通过第一曲线的线性回归对两条曲线的浓度进行了反对。定量限制（LOQ）定义为80％<精度<精度<120％的最低浓度点与高于平均值（空白）+3×SD（空白）相关的信号相关。

The multiplexing performance was evaluated by measuring the areas of the prm-PASEF traces for the 186 heavy AQUA peptides spiked at a constant concentration. The relative standard deviation (RSD) of the areas and the number of data points across the chromatographic peak (using the highest PRM transition) was calculated over the 30 prm-PASEF runs.

PRM-PASEF利用被困的离子迁移率（TIMS）设备可显着提高该方法的多路复用能力。这允许对目标采集进行大规模并行化，对快速LC和UHPLC的敏感性和更大的实施灵活性无效。TIMS单元的捕获和洗脱原理（图2）允许在100ms TIMS扫描事件中获取所有靶向肽，即在离子迁移率尺寸中顺序洗脱的所有靶向肽。此外，由于累积100ms的肽在5ms峰值上以5ms的峰为MS，因此聚焦效果会增加敏感性。

The elution times of the 216 precursors spread over 22 min on a 30 min gradient. In prm-PASEF the acquisitionwindows are three-dimensional and an isolation window must be defined according to chromatographic elution time, ion mobility, and the quadrupole isolation m/z. As we set the retention windows to 40 s per precursor, it generated substantial overlapping on the LC retention time dimension. However, the IMS dimension reduces the final acquisition window overlap as shown in Figure 5a. Precursor ions with overlapping acquisition windows, which can still be separated in the ion mobility dimension are measured within the same prm-PASEF frame (Figure 3).

The absolute quantitation potential of prm-PASEF was evaluated by studying heavy/light ratios for the 15 spiked-in peptides pairs. The combination of selectivity and sensitivity allowed for good signal detection, even at low concentrations (Figure 7). The detailed analysis of the ATVVYQGER heavy/light pair revealed that the signal response can be fitted by linear regression over a concentration factor of 2900 (from 17.2 to 50,000 amol injected column). Moreover, the back-calculation of the concentration for two of the triplicates, based on the calibration curve established with the first one, confirmed that the quantitation can be determined with a ± 20% accuracy at all concentrations (Table 1). The Limit of Quantification (LOQ) was defined as the lowest concentration point within 80% < accuracy < 120% associated with a signal higher than the mean (blanks) + 3X SD(blanks).

在此实验中，在每个PRM-PaseF框架（图5B）内测量平均4个靶向离子，而不会影响灵敏度或周期时间。当IMS和LC尺寸重叠几种化合物时，它们以连续的PaseF帧进行测量（图4）。平均而言，在PRM-PaseF循环中有4个不同的PRM-PaseF帧。在最高的前体密度下，每个MS循环最多使用10帧（图5C）。每个MS周期的帧数不会影响敏感性，而是定义色谱峰的数据点的数量。尽管如此，没有色谱峰的采样不足（图5D），每个色谱峰的数据点中位数为25，这表明可以使用更短的梯度进行实验。良好的峰采样和保留的灵敏度允许即使没有内标准化，也可以用正确的RSD测量所有峰区域（图6）。