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

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