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Comparison of MS2, synchronous precursor selection MS3, and real-time search MS3 methodologies for lung proteomes of hydrogen sulfide treated swine

文献类型： 外文期刊

作者： Fu, Qin 1 ; Liu, Zhen 2 ; Bhawal, Ruchika 1 ; Anderson, Elizabeth T. 1 ; Sherwood, Robert W. 1 ; Yang, Yong 4 ; Thannh 1 ;

作者机构： 1.Cornell Univ, Inst Biotechnol, Prote & Metabol Facil, 526 Campus Rd, Ithaca, NY 14853 USA

2.Chinese Acad Agr Sci, Inst Anim Sci, State Key Lab Anim Nutr, 2 West Yuanmingyuan Rd, Beijing 100193, Peoples R China

3.Univ Liege, Gembloux Agrobio Tech, Precis Livestock & Nutr Unit, Teaching & Res Ctr, Passage Deportes 2, B-5030 Gembloux, Belgium

4.USDA ARS, Robert W Holley Ctr Agr & Hlth, 538 Tower Rd, Ithaca, NY 14853 USA

关键词： Quantitative proteomics; Isobaric tandem mass tags; Synchronous precursor selection; MS3; Real-time search; Lung tissues

期刊名称：ANALYTICAL AND BIOANALYTICAL CHEMISTRY （ 影响因子：4.142； 五年影响因子：3.863 ）

ISSN： 1618-2642

年卷期：

页码：

收录情况： SCI

摘要： Tandem mass tags (TMTs) have increasingly become an attractive technique for global proteomics. However, its effectiveness for multiplexed quantitation by traditional tandem mass spectrometry (MS2) suffers from ratio distortion. Synchronous precursor selection (SPS) MS3 has been widely accepted for improved quantitation accuracy, but concurrently decreased proteome coverage. Recently, a Real-Time Search algorithm has been integrated with the SPS MS3 pipeline (RTS MS3) to provide accurate quantitation and improved depth of coverage. In this mechanistic study of the impact of exposure to hydrogen sulfide (H2S) on the respiration of swine, we used TMT-based comparative proteomics of lung tissues from control and H2S-treated subjects as a test case to evaluate traditional MS2, SPS MS3, and RTS MS3 acquisition methods on both the Orbitrap Fusion and Orbitrap Eclipse platforms. Comparison of the results obtained by the MS2 with those of SPS MS3 and RTS MS3 methods suggests that the MS3-driven quantitative strategies provided a more accurate global-scale quantitation; however, only RTS MS3 provided proteomic coverage that rivaled that of traditional MS2 analysis. RTS MS3 not only yields more productive MS3 spectra than SPS MS3 but also appears to focus the analysis more effectively on unique peptides. Furthermore, pathway enrichment analyses of the H2S-altered proteins demonstrated that an additional apoptosis pathway was discovered exclusively by RTS MS3. This finding was verified by RT-qPCR, western blotting, and TUNEL staining experiments. We conclude that RTS MS3 workflow enables simultaneous improvement of quantitative accuracy and proteome coverage over alternative approaches (MS2 and SPS MS3).

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