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Protocol for using fluorescent sensors targeted to endogenous proteins (FluoSTEPs) to measure microdomain-specific signaling events.

ABSTRACT: Fluorescence-based sensors are powerful molecular tools for studying the spatiotemporal regulation of cell signaling, which is often organized into discrete microdomains. Here, we present a protocol for using fluorescent sensors targeted to endogenous proteins (FluoSTEPs), a new class of fluorescent sensors in which the functional probe is exclusively reconstituted at an endogenously expressed protein of interest associated with a specific microdomain. FluoSTEPs allow microdomain-specific signaling activities to be measured with high selectivity without perturbing the native stoichiometry of signaling components. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2020) and Tenner et al. (2021).

SUBMITTER: Zhang JZ

PROVIDER: S-EPMC8387570 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Protocol for using fluorescent sensors targeted to endogenous proteins (FluoSTEPs) to measure microdomain-specific signaling events.

Zhang Jason Z JZ Tenner Brian B Mehta Sohum S Zhang Jin J

STAR protocols 20210802 3

Fluorescence-based sensors are powerful molecular tools for studying the spatiotemporal regulation of cell signaling, which is often organized into discrete microdomains. Here, we present a protocol for using fluorescent sensors targeted to endogenous proteins (FluoSTEPs), a new class of fluorescent sensors in which the functional probe is exclusively reconstituted at an endogenously expressed protein of interest associated with a specific microdomain. FluoSTEPs allow microdomain-specific signal ...[more]

PMID: 34467224

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Project description:BackgroundBoth 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.ResultsHere 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.ConclusionsThis 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.

Dataset Information

Protocol for using fluorescent sensors targeted to endogenous proteins (FluoSTEPs) to measure microdomain-specific signaling events.

Publications

Protocol for using fluorescent sensors targeted to endogenous proteins (FluoSTEPs) to measure microdomain-specific signaling events.

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