Targeted mass spectrometry of endogenous LINE-1 proteins and ORF2p interactomics

ABSTRACT: Expression and activity of LINE-1 (L1) retrotransposons is known to occur in numerous cell-types and is 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 protein over-expression contexts and to indirect detection of endogenous ORF2p enzymatic activity, such as by the sequencing of de novo genomic insertions. Here we present targeted mass spectrometry assays, selected and parallel reaction monitoring (SRM and PRM, respectively) to detect and quantify L1 ORF1p and ORF2p in endogenous expression contexts. 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.

INSTRUMENT(S): TSQ Altis, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Colon, Oocyte

DISEASE(S): Colon Cancer,Malignant Neoplasm Of Ovary

SUBMITTER: Mathias Nielsen  

LAB HEAD: John LaCava

PROVIDER: PXD057269 | Pride | 2025-02-06

REPOSITORIES: pride