Hücre Hatlarında Endojen Proteinlerin Kantitatif Çoklu Hızlı İmmünopresipitasyon ve Kütle Spektrometrisi (qPLEX-RIME)

Özet (abstract)

Abstract Quantitative multiPLEXed Rapid Immunoprecipitation and Mass spectrometry of Endogenous proteins (qPLEX‐RIME) is a method for the quantitative discovery of endogenous protein complexes, without the need for genetically encoded affinity tags. It is particularly well suited to chromatin‐associated proteins and transcription factors, permitting rapid, robust, and quantitative identification of the protein components of a specifically purified complex directly from the endogenous protein of interest. The method uses sequential double fixation with disuccinimidyl glutarate (DSG) and formaldehyde to stabilize protein complexes and preserve low‐abundance or transient interactions. Target protein complexes are captured using magnetic‐bead‐coupled antibodies raised against the endogenous protein, and the resulting immunoprecipitates are washed extensively before on‐bead tryptic digestion. Resulting peptides from each experimental condition are labeled with isobaric tandem mass tags (TMT or TMTpro), pooled, and analyzed together in a single LC‐MS/MS run, allowing relative quantification of factor‐associated proteins across multiple conditions and biological replicates within one experiment. We further describe a complete bioinformatic workflow, using the qPLEXanalyzer R package, for processing raw peptide intensities into a statistically supported list of differentially enriched interacting proteins. Although the method depends on access to high‐resolution Tribrid mass spectrometry instrumentation and a highly specific antibody against the protein of interest, it represents a comparatively rapid and reproducible approach for the discovery of protein‐protein interactions, applicable across cell line models as well as patient‐derived xenograft and primary tumor tissue. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Sample processing and immunoprecipitation Basic Protocol 2 : Proteomic processing Basic Protocol 3 : Bioinformatic analysis

Yazarların özeti; kaynağından alınmıştır. Current Protocols, 2026 · DOI ↗