Targeted detection of endogenous LINE-1 proteins and ORF2p interactions
Abstract Background Both the expression and activities of LINE-1 (L1) retrotransposons are known to occur in numerous cell-types and are implicated in pathobiological contexts such as aging-related inflammation, autoimmunity, and in cancers. L1s encode two proteins that are translated from bicistron...
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BMC
2025-02-01
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| Series: | Mobile DNA |
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| Online Access: | https://doi.org/10.1186/s13100-024-00339-4 |
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| author | Mathias I. Nielsen Justina C. Wolters Omar G. Rosas Bringas Hua Jiang Luciano H. Di Stefano Mehrnoosh Oghbaie Samira Hozeifi Mats J. Nitert Alienke van Pijkeren Marieke Smit Lars ter Morsche Apostolos Mourtzinos Vikram Deshpande Martin S. Taylor Brian T. Chait John LaCava |
| author_facet | Mathias I. Nielsen Justina C. Wolters Omar G. Rosas Bringas Hua Jiang Luciano H. Di Stefano Mehrnoosh Oghbaie Samira Hozeifi Mats J. Nitert Alienke van Pijkeren Marieke Smit Lars ter Morsche Apostolos Mourtzinos Vikram Deshpande Martin S. Taylor Brian T. Chait John LaCava |
| author_sort | Mathias I. Nielsen |
| collection | DOAJ |
| description | Abstract Background Both the expression and activities of LINE-1 (L1) retrotransposons are known to occur in numerous cell-types and are implicated in pathobiological contexts such as aging-related inflammation, autoimmunity, and in cancers. L1s encode two proteins that are translated from bicistronic transcripts. The translation product of ORF1 (ORF1p) has been robustly detected by immunoassays and shotgun mass spectrometry (MS). Yet, more sensitive detection methods would enhance the use of ORF1p as a clinical biomarker. In contrast, until now, no direct evidence of endogenous L1 ORF2 translation to protein (ORF2p) has been shown. Instead, assays for ORF2p have been limited to ectopic L1 ORF over-expression contexts and to indirect detection of endogenous ORF2p enzymatic activity, such as by the sequencing of de novo genomic insertions. Immunoassays for endogenous ORF2p have been problematic, producing apparent false positives due to cross-reactivities, and shotgun MS has not yielded reliable evidence of ORF2p peptides in biological samples. Results Here we present targeted mass spectrometry assays, selected and parallel reaction monitoring (SRM and PRM, respectively) to detect and quantify L1 ORF1p and ORF2p at their endogenous abundances. We were able to quantify ORF1p and ORF2p present in our samples down to a range in the low attomoles. Confident in our ability to affinity enrich ORF2p, we describe an interactome associated with endogenous ORF2-containing macromolecular assemblies. Conclusions This is the first assay to demonstrate sensitive and robust quantitation of endogenous ORF2p. The ability to assay ORF2p directly and quantitatively will improve our understanding of the developmental and diseased cell states where L1 expression and its activity naturally occur. The ability to simultaneously assay endogenous L1 ORF1p and ORF2p is an important step forward for L1 analytical biochemistry. Endogenous ORF2p interactomes can now be presented with confidence that ORF2p is among the enriched proteins. |
| format | Article |
| id | doaj-art-7d07637511d34b23aa2bba24c300b4c1 |
| institution | Kabale University |
| issn | 1759-8753 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | BMC |
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| series | Mobile DNA |
| spelling | doaj-art-7d07637511d34b23aa2bba24c300b4c12025-02-09T12:41:01ZengBMCMobile DNA1759-87532025-02-0116111910.1186/s13100-024-00339-4Targeted detection of endogenous LINE-1 proteins and ORF2p interactionsMathias I. Nielsen0Justina C. Wolters1Omar G. Rosas Bringas2Hua Jiang3Luciano H. Di Stefano4Mehrnoosh Oghbaie5Samira Hozeifi6Mats J. Nitert7Alienke van Pijkeren8Marieke Smit9Lars ter Morsche10Apostolos Mourtzinos11Vikram Deshpande12Martin S. Taylor13Brian T. Chait14John LaCava15Laboratory of Cellular and Structural Biology, The Rockefeller UniversityDepartment of Pediatrics, University Medical Center Groningen, University of GroningenEuropean Research Institute for the Biology of Ageing, University Medical Center Groningen, University of GroningenLaboratory of Cellular and Structural Biology, The Rockefeller UniversityEuropean Research Institute for the Biology of Ageing, University Medical Center Groningen, University of GroningenLaboratory of Cellular and Structural Biology, The Rockefeller UniversityEuropean Research Institute for the Biology of Ageing, University Medical Center Groningen, University of GroningenDepartment of Pediatrics, University Medical Center Groningen, University of GroningenDepartment of Pediatrics, University Medical Center Groningen, University of GroningenDepartment of Pediatrics, University Medical Center Groningen, University of GroningenLaboratory of Cellular and Structural Biology, The Rockefeller UniversityLaboratory of Cellular and Structural Biology, The Rockefeller UniversityDepartment of Pathology, Mass General Brigham and Harvard Medical SchoolDepartment of Pathology, Mass General Brigham and Harvard Medical SchoolLaboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller UniversityLaboratory of Cellular and Structural Biology, The Rockefeller UniversityAbstract Background Both the expression and activities of LINE-1 (L1) retrotransposons are known to occur in numerous cell-types and are implicated in pathobiological contexts such as aging-related inflammation, autoimmunity, and in cancers. L1s encode two proteins that are translated from bicistronic transcripts. The translation product of ORF1 (ORF1p) has been robustly detected by immunoassays and shotgun mass spectrometry (MS). Yet, more sensitive detection methods would enhance the use of ORF1p as a clinical biomarker. In contrast, until now, no direct evidence of endogenous L1 ORF2 translation to protein (ORF2p) has been shown. Instead, assays for ORF2p have been limited to ectopic L1 ORF over-expression contexts and to indirect detection of endogenous ORF2p enzymatic activity, such as by the sequencing of de novo genomic insertions. Immunoassays for endogenous ORF2p have been problematic, producing apparent false positives due to cross-reactivities, and shotgun MS has not yielded reliable evidence of ORF2p peptides in biological samples. Results Here we present targeted mass spectrometry assays, selected and parallel reaction monitoring (SRM and PRM, respectively) to detect and quantify L1 ORF1p and ORF2p at their endogenous abundances. We were able to quantify ORF1p and ORF2p present in our samples down to a range in the low attomoles. Confident in our ability to affinity enrich ORF2p, we describe an interactome associated with endogenous ORF2-containing macromolecular assemblies. Conclusions This is the first assay to demonstrate sensitive and robust quantitation of endogenous ORF2p. The ability to assay ORF2p directly and quantitatively will improve our understanding of the developmental and diseased cell states where L1 expression and its activity naturally occur. The ability to simultaneously assay endogenous L1 ORF1p and ORF2p is an important step forward for L1 analytical biochemistry. Endogenous ORF2p interactomes can now be presented with confidence that ORF2p is among the enriched proteins.https://doi.org/10.1186/s13100-024-00339-4Targeted proteomicsMass spectrometrySRMPRMLINE-1Retrotransposon |
| spellingShingle | Mathias I. Nielsen Justina C. Wolters Omar G. Rosas Bringas Hua Jiang Luciano H. Di Stefano Mehrnoosh Oghbaie Samira Hozeifi Mats J. Nitert Alienke van Pijkeren Marieke Smit Lars ter Morsche Apostolos Mourtzinos Vikram Deshpande Martin S. Taylor Brian T. Chait John LaCava Targeted detection of endogenous LINE-1 proteins and ORF2p interactions Mobile DNA Targeted proteomics Mass spectrometry SRM PRM LINE-1 Retrotransposon |
| title | Targeted detection of endogenous LINE-1 proteins and ORF2p interactions |
| title_full | Targeted detection of endogenous LINE-1 proteins and ORF2p interactions |
| title_fullStr | Targeted detection of endogenous LINE-1 proteins and ORF2p interactions |
| title_full_unstemmed | Targeted detection of endogenous LINE-1 proteins and ORF2p interactions |
| title_short | Targeted detection of endogenous LINE-1 proteins and ORF2p interactions |
| title_sort | targeted detection of endogenous line 1 proteins and orf2p interactions |
| topic | Targeted proteomics Mass spectrometry SRM PRM LINE-1 Retrotransposon |
| url | https://doi.org/10.1186/s13100-024-00339-4 |
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