Targeted detection of endogenous LINE-1 proteins and ORF2p interactions

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

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.

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.