Gas-phase fractionation DDA promotes in-depth DIA phosphoproteome analysis
Data-independent acquisition (DIA) is a promising method for quantitative proteomics. Library-based DIA database searching against project-specific data-dependent acquisition (DDA) spectral libraries is the gold standard. These libraries are constructed using material-consuming pre-fractionation two...
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Elsevier
2025-01-01
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844025003081 |
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| author | Zhiwei Tu Yabin Li Shuhui Ji Shanshan Wang Rui Zhou Gertjan Kramer Yu Cui Fei Xie |
| author_facet | Zhiwei Tu Yabin Li Shuhui Ji Shanshan Wang Rui Zhou Gertjan Kramer Yu Cui Fei Xie |
| author_sort | Zhiwei Tu |
| collection | DOAJ |
| description | Data-independent acquisition (DIA) is a promising method for quantitative proteomics. Library-based DIA database searching against project-specific data-dependent acquisition (DDA) spectral libraries is the gold standard. These libraries are constructed using material-consuming pre-fractionation two dimensional DDA analysis. The alternative to this is library-free DIA analysis. Limited sample amounts restrict the use of fractionation to build spectral libraries for post-translational modifications (PTMs) DIA analysis. We present the use of gas-phase fractionation (GPF) DDA data to improve the depth of library-free DIA identification for the phosphoproteome, called GPF-DDA hybrid DIA. This method fully utilizes the remnants of samples post-DIA analysis and leverages both library-based and -free DIA database searching. GPF-DDA hybrid DIA analyzes phosphopeptides surplus sample after DIA analysis using a number of DDA injections with each scanning different mass-to-charge (m/z) windows, instead of preforming traditional off-line fractionation-based DDA. The GPF-DDA data is integrated into the library-free DIA database search to create a hybrid library, enhancing phosphopeptide identification. Two GPF-DDA injections proved to increase 18 % phosphopeptide and 13 % phosphosite identification in HEK293 cell lines, while five injections resulted in up to 28 % phosphopeptide and 21 % phosphosite increases compared to library-free DIA analysis alone. We used GPF-DDA hybrid DIA phosphoproteomics to characterize lung tissue upon direct (smoke induced) and indirect (sepsis induced) acute lung injury (ALI) in mice. The differentially expressed phosphosites (DEPsites) in direct ALI were found in proteins related to mRNA processing and RNA. DEPsites in indirect ALI were enriched in proteins related to microtubule polymerization, positive regulation of microtubule polymerization and fibroblast migration. This study demonstrates that GPF-DDA hybrid DIA analysis workflow can indeed promote depth of DIA analysis of phosphoproteome and could be extended to DIA analysis of other PTMs. |
| format | Article |
| id | doaj-art-1ae555b34db14e06b8e59f98ff5c3925 |
| institution | Kabale University |
| issn | 2405-8440 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Heliyon |
| spelling | doaj-art-1ae555b34db14e06b8e59f98ff5c39252025-02-02T05:28:34ZengElsevierHeliyon2405-84402025-01-01112e41928Gas-phase fractionation DDA promotes in-depth DIA phosphoproteome analysisZhiwei Tu0Yabin Li1Shuhui Ji2Shanshan Wang3Rui Zhou4Gertjan Kramer5Yu Cui6Fei Xie7State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, 102206, Beijing, ChinaCollege of Pulmonary and Critical Care Medicine, Chinese PLA General Hospital, 100048, Beijing, ChinaState Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, 102206, Beijing, ChinaState Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, 102206, Beijing, ChinaThe First Affiliated Hospital of Henan University of Chinese Medicine, 450000, Zhengzhou, Henan, ChinaLaboratory for Mass Spectrometry of Biomolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, the NetherlandsState Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, 102206, Beijing, China; Corresponding author.College of Pulmonary and Critical Care Medicine, Chinese PLA General Hospital, 100048, Beijing, China; Corresponding author.Data-independent acquisition (DIA) is a promising method for quantitative proteomics. Library-based DIA database searching against project-specific data-dependent acquisition (DDA) spectral libraries is the gold standard. These libraries are constructed using material-consuming pre-fractionation two dimensional DDA analysis. The alternative to this is library-free DIA analysis. Limited sample amounts restrict the use of fractionation to build spectral libraries for post-translational modifications (PTMs) DIA analysis. We present the use of gas-phase fractionation (GPF) DDA data to improve the depth of library-free DIA identification for the phosphoproteome, called GPF-DDA hybrid DIA. This method fully utilizes the remnants of samples post-DIA analysis and leverages both library-based and -free DIA database searching. GPF-DDA hybrid DIA analyzes phosphopeptides surplus sample after DIA analysis using a number of DDA injections with each scanning different mass-to-charge (m/z) windows, instead of preforming traditional off-line fractionation-based DDA. The GPF-DDA data is integrated into the library-free DIA database search to create a hybrid library, enhancing phosphopeptide identification. Two GPF-DDA injections proved to increase 18 % phosphopeptide and 13 % phosphosite identification in HEK293 cell lines, while five injections resulted in up to 28 % phosphopeptide and 21 % phosphosite increases compared to library-free DIA analysis alone. We used GPF-DDA hybrid DIA phosphoproteomics to characterize lung tissue upon direct (smoke induced) and indirect (sepsis induced) acute lung injury (ALI) in mice. The differentially expressed phosphosites (DEPsites) in direct ALI were found in proteins related to mRNA processing and RNA. DEPsites in indirect ALI were enriched in proteins related to microtubule polymerization, positive regulation of microtubule polymerization and fibroblast migration. This study demonstrates that GPF-DDA hybrid DIA analysis workflow can indeed promote depth of DIA analysis of phosphoproteome and could be extended to DIA analysis of other PTMs.http://www.sciencedirect.com/science/article/pii/S2405844025003081DIAPhosphoproteomeGas phase fractionationGPF DDAARDS/ALI |
| spellingShingle | Zhiwei Tu Yabin Li Shuhui Ji Shanshan Wang Rui Zhou Gertjan Kramer Yu Cui Fei Xie Gas-phase fractionation DDA promotes in-depth DIA phosphoproteome analysis Heliyon DIA Phosphoproteome Gas phase fractionation GPF DDA ARDS/ALI |
| title | Gas-phase fractionation DDA promotes in-depth DIA phosphoproteome analysis |
| title_full | Gas-phase fractionation DDA promotes in-depth DIA phosphoproteome analysis |
| title_fullStr | Gas-phase fractionation DDA promotes in-depth DIA phosphoproteome analysis |
| title_full_unstemmed | Gas-phase fractionation DDA promotes in-depth DIA phosphoproteome analysis |
| title_short | Gas-phase fractionation DDA promotes in-depth DIA phosphoproteome analysis |
| title_sort | gas phase fractionation dda promotes in depth dia phosphoproteome analysis |
| topic | DIA Phosphoproteome Gas phase fractionation GPF DDA ARDS/ALI |
| url | http://www.sciencedirect.com/science/article/pii/S2405844025003081 |
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