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

• Published in: Analytical and bioanalytical chemistry Vol. 413; no. 2; pp. 419 - 429
• Main Authors: Fu, Qin, Liu, Zhen, Bhawal, Ruchika, Anderson, Elizabeth T., Sherwood, Robert W., Yang, Yong, Thannhauser, Theodore, Schroyen, Martine, Tang, Xiangfang, Zhang, Hongfu, Zhang, Sheng
• Format: Journal Article Web Resource
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 2021; Springer Nature B.V; Springer
• Subjects: Agriculture & agronomie; Agriculture & agronomy; Analytical Chemistry; Apoptosis; Biochemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Food Science; Hydrogen sulfide; Laboratory Medicine; Life sciences; Livestock; Lungs; Mass spectrometry; Mass spectroscopy; Monitoring/Environmental Analysis; Peptides; Precursors; Proteomes; Proteomics; Quantitation; Real time; Research Paper; Sciences du vivant; Search algorithms; Swine; Western blotting; Workflow; Synchronous precursor selection; Lung tissues; Quantitative proteomics; Real-time search; Isobaric tandem mass tags; MS
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-020-03009-5

Tandem mass tags (TMTs) have increasingly become an attractive technique for global proteomics. However, its effectiveness for multiplexed quantitation by traditional tandem mass spectrometry (MS 2 ) suffers from ratio distortion. Synchronous precursor selection (SPS) MS 3 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 MS 3 pipeline (RTS MS 3 ) to provide accurate quantitation and improved depth of coverage. In this mechanistic study of the impact of exposure to hydrogen sulfide (H 2 S) on the respiration of swine, we used TMT-based comparative proteomics of lung tissues from control and H 2 S-treated subjects as a test case to evaluate traditional MS 2 , SPS MS 3 , and RTS MS 3 acquisition methods on both the Orbitrap Fusion and Orbitrap Eclipse platforms. Comparison of the results obtained by the MS 2 with those of SPS MS 3 and RTS MS 3 methods suggests that the MS 3 -driven quantitative strategies provided a more accurate global-scale quantitation; however, only RTS MS 3 provided proteomic coverage that rivaled that of traditional MS 2 analysis. RTS MS 3 not only yields more productive MS 3 spectra than SPS MS 3 but also appears to focus the analysis more effectively on unique peptides. Furthermore, pathway enrichment analyses of the H 2 S-altered proteins demonstrated that an additional apoptosis pathway was discovered exclusively by RTS MS 3 . This finding was verified by RT-qPCR, western blotting, and TUNEL staining experiments. We conclude that RTS MS 3 workflow enables simultaneous improvement of quantitative accuracy and proteome coverage over alternative approaches (MS 2 and SPS MS 3 ). Graphical abstract