In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies
Abstract Cross-linking mass spectrometry has evolved as a powerful technique to study protein-protein interactions and to provide structural information. Low reaction efficiencies, and complex matrices lead to challenging system wide crosslink analysis. We improved and streamlined an Azide-A-DSBSO b...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-08-01
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| Series: | Communications Chemistry |
| Online Access: | https://doi.org/10.1038/s42004-025-01644-6 |
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| author | Philipp Bräuer Laszlo Tirian Fränze Müller Karl Mechtler Manuel Matzinger |
| author_facet | Philipp Bräuer Laszlo Tirian Fränze Müller Karl Mechtler Manuel Matzinger |
| author_sort | Philipp Bräuer |
| collection | DOAJ |
| description | Abstract Cross-linking mass spectrometry has evolved as a powerful technique to study protein-protein interactions and to provide structural information. Low reaction efficiencies, and complex matrices lead to challenging system wide crosslink analysis. We improved and streamlined an Azide-A-DSBSO based in vivo crosslinking workflow employing two orthogonal effective enrichment steps: Affinity enrichment and size exclusion chromatography (SEC). Combined, they allow an effective enrichment of DSBSO containing peptides and remove the background of linear as well as mono-linked peptides. We found that the analysis of a single SEC fraction is effective to yield ~90% of all crosslinks, which is important whenever measurement time is limited, and sample throughput is crucial. Our workflow resulted in more than 5000 crosslinks from K562 cells and generated a comprehensive PPI network. From 393 PPI found within the nucleus, 56 are novel. We further show, that by applying DSBSO to nuclear extracts we yield more crosslinks on lower abundant proteins and showcase this on the DEAD-box RNA helicase DDX39B which is predominantly expressed in the nucleus. Our data indicates that DDX39B might be present in monomeric and dimeric forms together with DDX39A within the nuclear extracts analyzed. |
| format | Article |
| id | doaj-art-5d4fada6989340169ba173018a1e4b43 |
| institution | Kabale University |
| issn | 2399-3669 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Chemistry |
| spelling | doaj-art-5d4fada6989340169ba173018a1e4b432025-08-20T03:45:56ZengNature PortfolioCommunications Chemistry2399-36692025-08-018111310.1038/s42004-025-01644-6In vivo crosslinking and effective 2D enrichment for proteome wide interactome studiesPhilipp Bräuer0Laszlo Tirian1Fränze Müller2Karl Mechtler3Manuel Matzinger4Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)Institute of Molecular Biotechnology (IMBA), Austrian Academy of Sciences, Vienna BioCenter (VBC)Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)Abstract Cross-linking mass spectrometry has evolved as a powerful technique to study protein-protein interactions and to provide structural information. Low reaction efficiencies, and complex matrices lead to challenging system wide crosslink analysis. We improved and streamlined an Azide-A-DSBSO based in vivo crosslinking workflow employing two orthogonal effective enrichment steps: Affinity enrichment and size exclusion chromatography (SEC). Combined, they allow an effective enrichment of DSBSO containing peptides and remove the background of linear as well as mono-linked peptides. We found that the analysis of a single SEC fraction is effective to yield ~90% of all crosslinks, which is important whenever measurement time is limited, and sample throughput is crucial. Our workflow resulted in more than 5000 crosslinks from K562 cells and generated a comprehensive PPI network. From 393 PPI found within the nucleus, 56 are novel. We further show, that by applying DSBSO to nuclear extracts we yield more crosslinks on lower abundant proteins and showcase this on the DEAD-box RNA helicase DDX39B which is predominantly expressed in the nucleus. Our data indicates that DDX39B might be present in monomeric and dimeric forms together with DDX39A within the nuclear extracts analyzed.https://doi.org/10.1038/s42004-025-01644-6 |
| spellingShingle | Philipp Bräuer Laszlo Tirian Fränze Müller Karl Mechtler Manuel Matzinger In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies Communications Chemistry |
| title | In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies |
| title_full | In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies |
| title_fullStr | In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies |
| title_full_unstemmed | In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies |
| title_short | In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies |
| title_sort | in vivo crosslinking and effective 2d enrichment for proteome wide interactome studies |
| url | https://doi.org/10.1038/s42004-025-01644-6 |
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