Developing a new cleavable crosslinker reagent for in-cell crosslinking
Abstract Crosslinking mass spectrometry (XL-MS) is a powerful technology that recently emerged as an essential complementary tool for elucidating protein structures and mapping interactions within a protein network. Crosslinkers which are amenable to post-linking backbone cleavage simplify peptide i...
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Nature Portfolio
2025-06-01
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| Series: | Communications Chemistry |
| Online Access: | https://doi.org/10.1038/s42004-025-01568-1 |
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| author | Fränze Müller Bogdan R. Brutiu Iakovos Saridakis Thomas Leischner Micha J. Birklbauer Manuel Matzinger Mathias Madalinski Thomas Lendl Saad Shaaban Viktoria Dorfer Nuno Maulide Karl Mechtler |
| author_facet | Fränze Müller Bogdan R. Brutiu Iakovos Saridakis Thomas Leischner Micha J. Birklbauer Manuel Matzinger Mathias Madalinski Thomas Lendl Saad Shaaban Viktoria Dorfer Nuno Maulide Karl Mechtler |
| author_sort | Fränze Müller |
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| description | Abstract Crosslinking mass spectrometry (XL-MS) is a powerful technology that recently emerged as an essential complementary tool for elucidating protein structures and mapping interactions within a protein network. Crosslinkers which are amenable to post-linking backbone cleavage simplify peptide identification, aid in 3D structure determination and enable system-wide studies of protein-protein interactions (PPIs) in cellular environments. However, state-of-the-art cleavable linkers are fraught with practical limitations, including extensive evaluation of fragmentation energies and fragmentation behavior of the crosslinker backbone. We herein introduce DiSPASO (bis(2,5-dioxopyrrolidin-1-yl) 3,3’-((5-ethynyl-1,3-phenylene)bis(methylenesulfinyl))dipropanoate) as a lysine-selective, MS-cleavable crosslinker with an alkyne handle for affinity enrichment. DiSPASO was designed and developed for efficient cell membrane permeability and crosslinking while securing low cellular perturbation. We tested DiSPASO employing three different copper-based enrichment strategies using model systems with increasing complexity (Cas9-Halo, purified ribosomes, live cells). Fluorescence microscopy in-cell crosslinking experiments revealed a rapid uptake of DiSPASO into HEK 293 cells within 5 minutes. While DiSPASO represents progress in cellular PPI analysis, its limitations and low crosslinking yield in cellular environments require careful optimisation of the crosslinker design, highlighting the complexity of developing effective XL-MS tools and the importance of continuous innovation in accurately mapping PPI networks within dynamic cellular environments. |
| format | Article |
| id | doaj-art-daba8b097b604fa3b2c74bc697e91778 |
| institution | Kabale University |
| issn | 2399-3669 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Chemistry |
| spelling | doaj-art-daba8b097b604fa3b2c74bc697e917782025-08-20T03:27:09ZengNature PortfolioCommunications Chemistry2399-36692025-06-018111510.1038/s42004-025-01568-1Developing a new cleavable crosslinker reagent for in-cell crosslinkingFränze Müller0Bogdan R. Brutiu1Iakovos Saridakis2Thomas Leischner3Micha J. Birklbauer4Manuel Matzinger5Mathias Madalinski6Thomas Lendl7Saad Shaaban8Viktoria Dorfer9Nuno Maulide10Karl Mechtler11Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)Institute of Organic Chemistry, University of ViennaInstitute of Organic Chemistry, University of ViennaInstitute of Organic Chemistry, University of ViennaBioinformatics Research Group, University of Applied Sciences Upper AustriaInstitute of Molecular Pathology (IMP), Vienna BioCenter (VBC)Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)Institute of Organic Chemistry, University of ViennaBioinformatics Research Group, University of Applied Sciences Upper AustriaInstitute of Organic Chemistry, University of ViennaInstitute of Molecular Pathology (IMP), Vienna BioCenter (VBC)Abstract Crosslinking mass spectrometry (XL-MS) is a powerful technology that recently emerged as an essential complementary tool for elucidating protein structures and mapping interactions within a protein network. Crosslinkers which are amenable to post-linking backbone cleavage simplify peptide identification, aid in 3D structure determination and enable system-wide studies of protein-protein interactions (PPIs) in cellular environments. However, state-of-the-art cleavable linkers are fraught with practical limitations, including extensive evaluation of fragmentation energies and fragmentation behavior of the crosslinker backbone. We herein introduce DiSPASO (bis(2,5-dioxopyrrolidin-1-yl) 3,3’-((5-ethynyl-1,3-phenylene)bis(methylenesulfinyl))dipropanoate) as a lysine-selective, MS-cleavable crosslinker with an alkyne handle for affinity enrichment. DiSPASO was designed and developed for efficient cell membrane permeability and crosslinking while securing low cellular perturbation. We tested DiSPASO employing three different copper-based enrichment strategies using model systems with increasing complexity (Cas9-Halo, purified ribosomes, live cells). Fluorescence microscopy in-cell crosslinking experiments revealed a rapid uptake of DiSPASO into HEK 293 cells within 5 minutes. While DiSPASO represents progress in cellular PPI analysis, its limitations and low crosslinking yield in cellular environments require careful optimisation of the crosslinker design, highlighting the complexity of developing effective XL-MS tools and the importance of continuous innovation in accurately mapping PPI networks within dynamic cellular environments.https://doi.org/10.1038/s42004-025-01568-1 |
| spellingShingle | Fränze Müller Bogdan R. Brutiu Iakovos Saridakis Thomas Leischner Micha J. Birklbauer Manuel Matzinger Mathias Madalinski Thomas Lendl Saad Shaaban Viktoria Dorfer Nuno Maulide Karl Mechtler Developing a new cleavable crosslinker reagent for in-cell crosslinking Communications Chemistry |
| title | Developing a new cleavable crosslinker reagent for in-cell crosslinking |
| title_full | Developing a new cleavable crosslinker reagent for in-cell crosslinking |
| title_fullStr | Developing a new cleavable crosslinker reagent for in-cell crosslinking |
| title_full_unstemmed | Developing a new cleavable crosslinker reagent for in-cell crosslinking |
| title_short | Developing a new cleavable crosslinker reagent for in-cell crosslinking |
| title_sort | developing a new cleavable crosslinker reagent for in cell crosslinking |
| url | https://doi.org/10.1038/s42004-025-01568-1 |
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