Ardeljan D, Wang X, Oghbaie M, Taylor MS, Husband D, Deshpande V, Steranka JP, Gorbounov M, Yang WR, Sie B, Larman HB, Jiang H, Molloy KR, Altukhov I, Li Z, McKerrow W, Fenyö D, Burns KH, LaCava J. *Mobile DNA* · January 1, 2020 · DOI: [10.1186/s13100-019-0191-2](https://doi.org/10.1186/s13100-019-0191-2) How to cite ### AMA Ardeljan D, Wang X, Oghbaie M, et al. LINE-1 ORF2p expression is nearly imperceptible in human cancers. *Mob DNA*. 2020;11:1. doi:10.1186/s13100-019-0191-2 ### APA ``` Ardeljan, D., Wang, X., Oghbaie, M., Taylor, M. S., Husband, D., Deshpande, V., Steranka, J. P., Gorbounov, M., Yang, W. R., Sie, B., Larman, H. B., Jiang, H., Molloy, K. R., Altukhov, I., Li, Z., McKerrow, W., Fenyö, D., Burns, K. H., & LaCava, J. (2020). LINE-1 ORF2p expression is nearly imperceptible in human cancers. *Mobile DNA*, 11, 1. https://doi.org/10.1186/s13100-019-0191-2 ``` ### BibTeX ``` @article{ardeljan2020line1orf2p, title = {LINE-1 ORF2p expression is nearly imperceptible in human cancers}, author = {Ardeljan, Daniel and Wang, Xuya and Oghbaie, Mehrnoosh and Taylor, Martin S. and Husband, David and Deshpande, Vikram and Steranka, Jared P. and Gorbounov, Mikhail and Yang, Wan Rou and Sie, Brandon and Larman, H. Benjamin and Jiang, Hua and Molloy, Kelly R. and Altukhov, Ilya and Li, Zhi and McKerrow, Wilson and Feny{\"o}, David and Burns, Kathleen H. and LaCava, John}, journal = {Mobile DNA}, volume = {11}, pages = {1}, year = {2020}, doi = {10.1186/s13100-019-0191-2} } ``` Even in cancers where LINE-1 retrotransposition is unequivocally active and ORF1p is abundant, the catalytic ORF2p protein evades direct detection by mass spectrometry, immunohistochemistry, immunoprecipitation, and Western blot — including with five newly validated rabbit monoclonal antibodies. The authors triangulated three independent approaches across CPTAC tumor proteomes, primary colorectal cancer immunoprecipitates, and ectopic-expression cell systems and reached the same conclusion: in human tumors the two LINE-1-encoded proteins are far more uncoupled in expression than experimental cell systems had suggested. [ Read publication at Mobile DNA ](https://doi.org/10.1186/s13100-019-0191-2) In this publication: - Reanalysis of deep mass-spectrometry proteomes from 102 breast and 176 ovarian CPTAC tumors detected ORF1p widely but yielded only one ambiguous PSM for ORF2p. - Five newly validated rabbit monoclonal antibodies cleanly detect ORF2p when forcibly expressed from L1 transgenes — ruling out reagent failure — yet none detect endogenous ORF2p in tumor sections or lysates. - Affinity purification of ORF1p from primary colorectal cancers, with mass-spectrometry interactome readout, failed to recover ORF2p as an interactor. - Ectopic-expression experiments confirm an ORF1p:ORF2p stoichiometric imbalance greater than 30:1; in vivo uncoupling appears more extreme still. Mass-spectrometry data deposited at ProteomeXchange (PXD013743). The authors took three independent approaches: reanalyzing deep mass-spectrometry proteomes from 102 breast and 176 ovarian tumors collected by the Clinical Proteomics Tumor Analysis Consortium (CPTAC); generating five new rabbit monoclonal antibodies against bacterially expressed ORF2p fragments; and affinity-purifying ORF1p from primary colorectal cancers and looking for ORF2p as an interactor by mass spectrometry. ORF1p was readily detected by every method. ORF2p was not. The new antibodies do work — they cleanly detect ORF2p when it is forcibly expressed from L1 transgenes — so the negative result is not a reagent failure. Ectopic-expression experiments confirm an ORF1p:ORF2p stoichiometric imbalance greater than 30:1, and the in vivo uncoupling appears more extreme still. The data reframe the LINE-1 expression model: direct detection of endogenous ORF2p is the standard assumption behind much of LINE-1 cancer biology, yet across CPTAC breast and ovarian proteomes, primary colorectal tumors, and ectopic-expression cell systems the protein remains below the floor of orthogonal methods — even where ORF1p is robust and de novo somatic insertions are abundant. For cancer researchers using ORF2p IHC or Western signal as a biomarker, negative or weak signal in a tumor does not necessarily mean LINE-1 is inactive. The work also sets a high bar for antibody specificity in retrotransposon biology and recommends amplification approaches (proximity ligation, targeted mass spectrometry at the attomole scale) before drawing expression conclusions from conventional protein assays. 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