Department of Chemistry, Fudan University , Shanghai, 200433, People's Republic of China.
Anal Chem. 2017 Oct 3;89(19):10248-10255. doi: 10.1021/acs.analchem.7b01635. Epub 2017 Sep 19.
Data-independent acquisition (DIA) has recently emerged as a powerful quantitative approach for large-scale proteome quantification, providing a sensitive and reproducible alternative to data-dependent acquisition (DDA). However, lack of compatible multiplexed quantification methods is a bottleneck of DIA. To alleviate this challenge, we present a mass defect based four-plex data-independent acquisition strategy, termed "MdFDIA", for parallel analysis of four different protein samples in a DIA experiment without the additional complexity of tandem mass spectrometry (MS) spectra. MdFDIA is a hybrid approach that combines stable isotope labeling with amino acids in cell culture (SILAC) and dimethyl labeling. Briefly, the isotopes CN-lysine (+8.0142 Da, light) and D-lysine (+8.0512 Da, heavy) were metabolically embedded in different proteome samples during cell culture. Then, two CN-lysine and D-lysine labeled protein samples were digested with Lys-C, followed by in vitro labeling with light (2CDH, +34.06312 Da) and heavy (2CD, +34.06896 Da) dimethyl groups, respectively, producing four different pseudoisobaric labeled protein samples. The labeled samples were then equally mixed and analyzed by DIA. The subtle mass differences between the four labeled forms in MS scans can be resolved on an Orbitrap Fusion Lumos instrument to facilitate quantification without abundance information encoded in MS spectra. Additionally, a systematic investigation was carried out and revealed that MdFDIA enabled a significant decrease of the adverse impact on the accuracy of the quantitative assays arising from the chromatographic isotope effect, especially the deuterium effect, which typically occurs in a DDA experiment. Additionally, MdFDIA provided a method for validating the fragment type in the DIA spectra identification result. Furthermore, MdFDIA was applied to quantitative proteome analyses of four different breast cancer cell lines, demonstrating the feasibility of this strategy for biological applications.
数据非依赖性采集（DIA）最近成为一种强大的大规模蛋白质组定量方法，为数据依赖性采集（DDA）提供了一种敏感且可重复的替代方法。然而，缺乏兼容的多重定量方法是 DIA 的瓶颈。为了缓解这一挑战，我们提出了一种基于质量亏损的四plex 数据非依赖性采集策略，称为“MdFDIA”，用于在 DIA 实验中平行分析四种不同的蛋白质样品，而无需串联质谱（MS）谱的额外复杂性。MdFDIA 是一种混合方法，结合了稳定同位素标记与细胞培养中的氨基酸（SILAC）和二甲基标记。简而言之，同位素 CN-赖氨酸（+8.0142 Da，轻）和 D-赖氨酸（+8.0512 Da，重）在细胞培养过程中代谢嵌入到不同的蛋白质样品中。然后，用 Lys-C 消化两个 CN-赖氨酸和 D-赖氨酸标记的蛋白质样品，然后分别用轻（2CDH，+34.06312 Da）和重（2CD，+34.06896 Da）二甲基基团进行体外标记，产生四个不同的拟等压标记蛋白质样品。然后将标记的样品等量混合并通过 DIA 进行分析。在 Orbitrap Fusion Lumos 仪器上，MS 扫描中四种标记形式之间的微小质量差异可以得到解析，从而在没有 MS 谱中丰度信息编码的情况下促进定量。此外，进行了系统研究，结果表明，MdFDIA 显著降低了色谱同位素效应（特别是在 DDA 实验中通常发生的氘效应）对定量分析准确性的不利影响。此外，MdFDIA 提供了一种用于验证 DIA 谱识别结果中片段类型的方法。此外，MdFDIA 应用于四种不同乳腺癌细胞系的定量蛋白质组学分析，证明了该策略在生物学应用中的可行性。
