Laboratory for the Analysis of Medicines, Center for Interdisciplinary Research on Medicines (CIRM), ULiege, Quartier Hopital, Avenue Hippocrate 15, 4000, Liege, Belgium.
Laboratory for the Analysis of Medicines, Center for Interdisciplinary Research on Medicines (CIRM), ULiege, Quartier Hopital, Avenue Hippocrate 15, 4000, Liege, Belgium.
Talanta. 2020 Jun 1;213:120812. doi: 10.1016/j.talanta.2020.120812. Epub 2020 Feb 8.
Ion mobility (IM) mass spectrometry allows conducting data independent acquisition (DIA) where all ions entering the instrument are fragmented based on their drift time. In this work, DIA operational parameters were first optimized using a design of experiments. The optimization of data treatment involved a smoothing algorithm of the IM dimension, which increased the number of identified peptides. Then, classical DDA and IM-based DIA were compared injecting increasing amounts of a complex proteome digest (E. coli). Results revealed that compared to DDA, DIA allowed to identify from 2 to 3.3 times more proteins, depending on the injected quantity. To evaluate proteome coverage, endogenous proteins in E. coli cells were sorted by abundance deciles. A large majority of the proteins uniquely observed in DDA were part of the 10% most abundant protein groups. Interestingly, owing to the absence of ion-picking algorithm, DIA allowed to identify proteins coming from a broader concentration range therefore greatly improving proteome coverage. Furthermore, ion mobility separation improved coverage by separating co-eluting peptides. Physicochemical properties of peptides uniquely detected by DIA or DDA were also compared using supervised and unsupervised multivariate analysis. As a result, peptides having a higher mass and being relatively hydrophobic were significantly more identified in DIA. Finally, semi-quantitative performance of both methods was investigated and proved to be comparable, except that DIA demonstrated a better sensitivity than DDA. As a conclusion, we demonstrated in this study that both acquisition modes provide complementary information about the proteome under investigation.
离子淌度（IM）质谱允许进行数据非依赖性采集（DIA），其中所有进入仪器的离子都根据其漂移时间进行碎片化。在这项工作中，首先使用实验设计优化了 DIA 操作参数。数据处理的优化涉及 IM 维度的平滑算法，这增加了鉴定肽的数量。然后，比较了注入越来越多的复杂蛋白质组消化物（大肠杆菌）时，经典的 DDA 和基于 IM 的 DIA。结果表明，与 DDA 相比，DIA 允许根据注入量鉴定出 2 到 3.3 倍更多的蛋白质。为了评估蛋白质组覆盖率，通过丰度十分位数对大肠杆菌细胞中的内源性蛋白质进行排序。在 DDA 中唯一观察到的大多数蛋白质都属于 10%最丰富的蛋白质组。有趣的是，由于没有离子选择算法，DIA 允许鉴定来自更广泛浓度范围的蛋白质，从而大大提高了蛋白质组覆盖率。此外，离子淌度分离通过分离共洗脱肽来提高覆盖率。还使用有监督和无监督多元分析比较了 DIA 或 DDA 唯一检测到的肽的物理化学性质。结果表明，DIA 中鉴定出的肽具有更高的质量和相对疏水性。最后，研究了这两种方法的半定量性能，结果证明它们是可比的，除了 DIA 比 DDA 具有更好的灵敏度。总之，我们在这项研究中证明了这两种采集模式都提供了关于所研究蛋白质组的互补信息。
