An optimized co-immunoprecipitation protocol for the analysis of endogenous protein-protein interactions in cell lines using mass spectrometry

Lagundžin, Dragana; Krieger, Kimiko L.; Law, Henry C-H; Woods, Nicholas T.

doi:10.1016/j.xpro.2022.101234

Cited by 9 publications

(8 citation statements)

References 7 publications

(12 reference statements)

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“…Pierce Protein G Magnetic Beads (ThermoFisher, #88847) were pre-incubated with 70 ug DUNP19 antibody or hIgG1 mAb in PBS at room temperature (RT) for 1 h. Bead-antibody conjugate was recovered using magnetic separation before adding 300 ug protein lysates (isolated from U118MG cells) for 2 h on ice. Beads were washed with PBS and an on-bead digestion was performed with 0.25 % Trypsin for 16 h. MS-MS was run using the Agilent 6530 LC/MS in collaboration with the UCLA Molecular Instrumentation Center according to previously described protocols (33, 34).…”

Section: Methodsmentioning

confidence: 99%

Development of a LRRC15-Targeted Radio-Immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

Storey,

Altai,

Lückerath

et al. 2024

Preprint

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Leucine-rich repeat containing 15 (LRRC15) has emerged as an attractive biomarker and target for cancer therapy. We have developed a humanized monoclonal antibody (mAb), DUNP19, that specifically binds to a phylogenetically conserved LRRC15 epitope and is internalized by target-expressing cancer and stromal cells. In xenograft mouse models, Lutetium-177 labeled DUNP19 ([177Lu]-DUNP19) enables non-invasive imaging and precise radiotherapy to LRRC15-expressing cancer cells and murine cancer-associated fibroblasts (CAFs), halting tumor progression and prolonging survival with minimal toxicity. Transcriptomic analyses of [177Lu]-DUNP19-treated tumors reveal a loss of pro-tumorigenic mechanisms, including a transforming growth factor beta (TGFβ)-driven and LRRC15+ signature associated with immunotherapy resistance. Together, these results demonstrate that radio-theranostic targeting of LRRC15 with DUNP19 is a compelling precision medicine platform for image-guided diagnosis, eradication, and reprogramming of LRRC15+ tumor tissue that drives immuno-resistance and aggressive disease.SIGNIFICANCEWe introduce a pioneering LRRC15-guided radio-theranostic approach integrating clinical imaging and radioimmunotherapy. Our strategy utilizes a mAb, DUNP19, to target LRRC15-expressing cancer cells and fibroblasts, demonstrating significant tumor reduction, prolonged survival, and reversal of TGFβ-driven treatment resistance. This approach offers a promising strategy for improving outcomes in aggressive cancers.

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Section: Methodsmentioning

confidence: 99%

Development of a LRRC15-Targeted Radio-Immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

Storey,

Altai,

Lückerath

et al. 2024

Preprint

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show abstract

“…80 µL of Anti-FLAG M2 affinity gel per sample were washed twice in IP lysis buffer and incubated overnight with pre-cleared lysates. The day after, beads were washed twice with Wash buffer A (10 mM HEPES, pH 7.4, 10mM KCl, 10mM NaCl, 1 mM MgCl2, 0.05% Nonidet P-40) once with Wash buffer B (10 mM HEPES, pH 7.4, 10mM KCl, 0.07% Nonidet P-40), as described by Lagundzin, D. et al 54 , and two times with PBS. Beads were further processed for Mass spectrometry as described below.…”

Section: Methodsmentioning

confidence: 99%

“…Enrichment of proteins, core and modified histones and transcription factors at the HIV-1 LTR was assessed by quantitative PCR using primers spanning the full promoter (Table 1) with GoTaq qPCR Master mix kit (Promega) in a CFX Connect Real-Time PCR thermocycler (BioRad). Relative enrichment over the DNA input was calculated with the 2 -ΔCt method 53 .…”

Section: Chromatin Immunoprecipitation (Chip) -Qpcrmentioning

confidence: 99%

PCID2 dysregulates transcription and viral RNA processing to promote HIV-1 latency

Crespo,

Ne,

Reinders

et al. 2023

Preprint

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HIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. To elucidate the molecular players that govern latency, we previously performed a dCas9-chromatin immunoprecipitation coupled with mass spectrometry (Catchet-MS) and identified the interactome of the latent HIV-1 LTR. Here we characterize the Catchet-MS-identified PCI domain-containing 2 (PCID2) protein, a component of the TREX2 complex, to play a dual role in promoting HIV-1 latency by enforcing both transcriptional repression and post-transcriptional blocks to HIV-1 gene expression. PCID2 bound the latent HIV-1 LTR and repressed transcription initiation during latency. Depletion of PCID2 remodelled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and reversal of latency. Immunoprecipitation coupled to Mass Spectrometry identified PCID2-interacting proteins to include members of the spliceosome, including negative viral RNA (vRNA) alternative splicing regulators, and PCID2 depletion resulted in over-splicing of intron-containing vRNA and misregulated expression of vRNA splice variants. We demonstrate that MCM3AP and DSS1, two other RNA-binding TREX2 complex subunits that comprise the dock of the complex also inhibit transcription initiation and viral RNA alternative splicing during latency and similarly to PCID2 function as prominent latency associated repressors of HIV-1 gene expression. Thus, PCID2 is a novel HIV-1 latency-promoting factor, which in context of the TREX2 sub-complex PCID2-DSS1-MCM3AP blocks transcription and dysregulates vRNA processing.

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“…As one example, ~5,700 proteins and over 27000 complex PPIs were identified using LC-MS/MS and merged into a protein-protein interactome, termed BraInMap ( 238 ). Lastly, it is worth to mention co-immunoprecipitation (coIP-MS) followed by mass spectrometry, which is a common practice to interrogate proteins interacting with a given protein bait ( 44 ).…”

Section: Technology-based Omicsmentioning

confidence: 99%

Advances and Trends in Omics Technology Development

2022

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The human history has witnessed the rapid development of technologies such as high-throughput sequencing and mass spectrometry that led to the concept of “omics” and methodological advancement in systematically interrogating a cellular system. Yet, the ever-growing types of molecules and regulatory mechanisms being discovered have been persistently transforming our understandings on the cellular machinery. This renders cell omics seemingly, like the universe, expand with no limit and our goal toward the complete harness of the cellular system merely impossible. Therefore, it is imperative to review what has been done and is being done to predict what can be done toward the translation of omics information to disease control with minimal cell perturbation. With a focus on the “four big omics,” i.e., genomics, transcriptomics, proteomics, metabolomics, we delineate hierarchies of these omics together with their epiomics and interactomics, and review technologies developed for interrogation. We predict, among others, redoxomics as an emerging omics layer that views cell decision toward the physiological or pathological state as a fine-tuned redox balance.

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An optimized co-immunoprecipitation protocol for the analysis of endogenous protein-protein interactions in cell lines using mass spectrometry

Cited by 9 publications

References 7 publications

Development of a LRRC15-Targeted Radio-Immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

Development of a LRRC15-Targeted Radio-Immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

PCID2 dysregulates transcription and viral RNA processing to promote HIV-1 latency

Advances and Trends in Omics Technology Development

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