Immunoprecipitation Followed By Mass Spectrometry Research Articles

Immunoprecipitation Followed by Mass Spectrometry: An Approach for Identifying Host-Viral Protein-Protein Interactions.

As obligate intracellular parasites, viruses rely on the efficient manipulation of the cell they invade in order to multiply and spread. Protein-protein interactions between viral proteins (or their complexes) and cellular proteins are at the interface between virus and host and hence crucial for the outcome of the infection. Multiple techniques can be used to study protein-protein interactions in vivo in the context of the infected cell; among them, immunoprecipitation followed by mass spectrometry (IP-MS) has proven an efficient approach for the unbiased identification of protein complexes containing a viral protein of interest. In this chapter, we discuss how to employ IP-MS to define the interactome of plant virus proteins by using transient expression in the experimental host Nicotiana benthamiana, using the geminivirus tomato yellow leaf curl virus (TYLCV) as an example.

A ZTF-7/RPS-2 complex mediates the cold-warm response in C. elegans.

Temperature greatly affects numerous biological processes in all organisms. How multicellular organisms respond to and are impacted by hypothermic stress remains elusive. Here, we found that cold-warm stimuli induced depletion of the RNA exosome complex in the nucleoli but enriched it in the nucleoplasm. To further understand the function and mechanism of cold-warm stimuli, we conducted forward genetic screening and identified ZTF-7, which is required for RNA exosome depletion from nucleoli upon transient cold-warm exposure in C. elegans. ZTF-7 is a putative ortholog of human ZNF277 that may contribute to language impairments. Immunoprecipitation followed by mass spectrometry (IP-MS) found that ZTF-7 interacted with RPS-2, which is a ribosomal protein of the small subunit and participates in pre-rRNA processing. A partial depletion of RPS-2 and other proteins of the small ribosomal subunit blocked the cold-warm stimuli-induced reduction of exosome subunits from the nucleoli. These results established a novel mechanism by which C. elegans responds to environmental cold-warm exposure.

The novel activity of Argonautes in intron splicing: A transcriptome-wide survey in plants

The importance of the evolutionarily conserved Argonaute (AGO) proteins has been well recognized for their involvement in the RNA interference pathways. Recent discoveries in animals demonstrated that AGOs also participate in alternative splicing (AS). Motivated by the question whether the AGO proteins are also functional in RNA splicing in plants, we searched for the introns excised through an AGO-dependent manner in Arabidopsis (Arabidopsis thaliana). RNA sequencing (RNA-seq) data analysis uncovered hundreds of the introns up- or down-regulated in the ago1 and ago4 mutants, respectively. For different genes, AGOs might play either a positive or a negative role in intron excision, which was further validated by reverse transcription-polymerase chain reaction (RT-PCR). Some introns were specifically regulated by one of the AGO proteins, while some were regulated by both AGOs. Besides, a large portion of the AGO-dependent introns were organ-specifically regulated. RNA immunoprecipitation combined with high-throughput sequencing (RIP-seq) revealed that both AGOs preferentially bound to the intronic regions, supporting their high intron binding affinities. Immunoprecipitation followed by mass spectrometry (IP-MS) was performed to identify the proteins potentially interacting with the two AGOs. Six novel interactors (two interacting with AGO1 and four with both AGOs) involved in mRNA binding were uncovered, which might facilitate AGO–intron recognition. Analysis of the RNA-seq data from the rice (Oryza sativa) ago18 mutants revealed that hundreds of the introns were expressed in an AGO18-dependent manner. In summary, our results point to the novel role of the plant AGOs in intron splicing, paving a way for further studies on the mechanisms underlying AGO-mediated RNA splicing.

Genome-wide CRISPR/Cas9 library screen identifies PCMT1 as a critical driver of ovarian cancer metastasis

BackgroundThe development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance.MethodsGenome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging.ResultsWe found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors.ConclusionsThrough systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.

CDE-1 suppresses the production of risiRNA by coupling polyuridylation and degradation of rRNA

BackgroundModification of RNAs, particularly at the terminals, is critical for various essential cell processes; for example, uridylation is implicated in tumorigenesis, proliferation, stem cell maintenance, and immune defense against viruses and retrotransposons. Ribosomal RNAs can be regulated by antisense ribosomal siRNAs (risiRNAs), which downregulate pre-rRNAs through the nuclear RNAi pathway in Caenorhabditis elegans. However, the biogenesis and regulation of risiRNAs remain obscure. Previously, we showed that 26S rRNAs are uridylated at the 3′-ends by an unknown terminal polyuridylation polymerase before the rRNAs are degraded by a 3′ to 5′ exoribonuclease SUSI-1(ceDIS3L2).ResultsHere, we found that CDE-1, one of the three C.elegans polyuridylation polymerases (PUPs), is specifically involved in suppressing risiRNA production. CDE-1 localizes to perinuclear granules in the germline and uridylates Argonaute-associated 22G-RNAs, 26S, and 5.8S rRNAs at the 3′-ends. Immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CDE-1 interacts with SUSI-1(ceDIS3L2). Consistent with these results, both CDE-1 and SUSI-1(ceDIS3L2) are required for the inheritance of RNAi.ConclusionsThis work identified a rRNA surveillance machinery of rRNAs that couples terminal polyuridylation and degradation.

A high-throughput pipeline for validation of antibodies.

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).

Abstract 1841: Enrichment of IGF1R-AKT-mTOR pathway proteins using immunoprecipitation and proteomic analysis by mass spectrometry

Abstract Background: A major bottleneck in the proteomic characterization of signaling pathway targets is the lack of methods/reagents to quantify medium to low levels of proteins of interest in human samples. Immunoprecipitation followed by mass spectrometry (IP-MS) enables the simultaneous analysis of protein expression, protein-protein interactions and post-translational modifications (PTMs). Immunoprecipitation provides both enrichment and increased sensitivity while the MS provides specific identification, high selectivity and multiplex possibility. Dysregulation of IGF1R -PI3K-AKT-mTOR signaling is a pivotal driver for many cancers making this pathway an attractive therapeutic target for cancer therapy. The quantitative evaluation of protein expression and PTM status of these signal transduction proteins will aid in the precise characterization of the disease at the diagnostic or prognostic level and to monitor its progression and treatment response. Methods: To investigate the IGF1R-PI3K-AKT-mTOR signaling pathway, we used stable isotopic amino acids in cell culture (SILAC) to differentially label proteins for quantification in insulin growth factor (IGF) stimulated versus unstimulated A549 and HCT116 cells. Targets were immunoprecipitated from the cell lysates, captured on streptavidin or Protein A/G magnetic beads, and eluted from the beads for direct in-solution tryptic digestion. Quantitative changes in total and phosphorylated forms of AKT-mTOR pathway targets were evaluated by liquid chromatography-mass spectrometry (LC-MS) on a Thermo Scientific Orbitrap Fusion platform. Results: The optimized IP-MS protocol resulted in the identification of low copy number proteins that could not be detected by LC-MS analysis without enrichment. Some of the low abundance targets observed after enrichment included PI3K, PTEN, and AKT isoforms present at low to sub nanogram levels. Interacting proteins including the catalytic and regulatory subunits of PI3K were also identified indicating that the workflow supports co-immunoprecipitation of protein complexes. Enrichment of several IGF1R-PI3K-AKT-mTOR pathway targets (IGF1R, PI3KCA, PIK3R2, AKT, and PTEN) from two cell lysates facilitated the relative quantification of total and phosphorylated forms of these proteins by LC-MS analysis. Conclusion: SILAC combined with the improved IP-MS method permits relative quantification of specific and functional protein-protein interactions and PTMs in the IGF1R-PI3K-AKT-mTOR pathway, which can ultimately lead to better cancer characterization and monitoring. Citation Format: Suzanne Smith, Bhavin Patel, Ryan Bomgarden, Kay Opperman, John Rogers, Barbara Kaboord. Enrichment of IGF1R-AKT-mTOR pathway proteins using immunoprecipitation and proteomic analysis by mass spectrometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1841. doi:10.1158/1538-7445.AM2015-1841

Abstract 1615: Enrichment of EGFR/PI3K/AKT/PTEN proteins using immunoprecipitation and analysis with mass spectrometry-based proteomics

Abstract Background: The dynamic range of protein concentrations spans more than seven orders of magnitude in human cell lines and eleven orders of magnitude in human plasma/serum and tissue. Currently, a major bottleneck in the verification of candidate cancer biomarkers is the lack of methods/reagents to quantify medium to low levels of proteins of interest in human samples. Immunoprecipitation followed by mass spectrometry (IP-MS) allows for the simultaneous analysis of protein expression, protein-protein interactions and post-translational modifications (PTMs) of protein biomarkers. The immunoaffinity aspect provides both enrichment and increased sensitivity while the mass spectrometry provides high specificity, high selectivity and multiplex possibility. Dysregulated EGFR-PI3K-AKT signaling is considered pivotal for many cancers, and this pathway is a potential candidate for therapeutic targeting. The quantitative evaluation of protein expression and PTM status of these and other signal transduction protein targets in cell lines, tissue, and plasma will enable precise characterization of the disease at the diagnostic or prognostic level and to monitor its progression and treatment response. Methods: Here we evaluated immunoprecipitation with directly coupled antibody or biotinylated antibody with immobilized streptavidin resin for MS applications. We evaluated several beads/resins to precipitate EGFR from A431 cell lysate with subsequent detection and quantitation by mass spectrometry. EGFR, PI3K, AKT isoforms and PTEN were immunoprecipitated from two cell lysates using the optimized IP to MS workflow. A multiplex targeted assay (LC-SRM/MS) was developed and implemented to measure limit of quantitation (LLOQ) of the EGFR, PI3K, AKT2, AKT1, and PTEN tryptic peptides in two cell lysates and plasma. Results: Immunoprecipitation using magnetic beads resulted in overall higher yield of target protein and less non-specific binding for MS applications. Enrichment of EGFR, AKT isoforms, and PTEN in two cell lysates permitted detection by discovery MS and quantitation by targeted MS. Immunoprecipitation of EGFR and AKT2 resulted in simultaneous analysis of multiple phosphorylation sites. EGFR, AKT1, AKT2 and PTEN were quantitated in low nanogram range by LC-SRM/MS in two cell lysates. Enrichment of as low as 7ng/mL recombinant EGFR in human plasma matrix allowed absolute quantitation by targeted MS. Conclusion: The application of IP to the analysis of EGFR, PI3K, AKT and PTEN protein targets permits detection and quantitation of protein and PTMs at sub to low ng/mL concentrations with mass spectrometry approaches. Citation Format: Bhavinkumar Patel, Scott Meier, Kay Opperman, Paul Haney, Barbara Kaboord, John Rogers. Enrichment of EGFR/PI3K/AKT/PTEN proteins using immunoprecipitation and analysis with mass spectrometry-based proteomics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1615. doi:10.1158/1538-7445.AM2014-1615

Chrebp regulates the transcriptional activity of androgen receptor in prostate cancer.

Androgen receptor (AR), a member of nuclear hormone receptor, plays an essential role in the initiation and progression of prostate cancer (PCa). In the present study, by way of immunoprecipitation followed by mass spectrometry (IP/MS) system, we found that carbohydrate-responsive element-binding protein (Chrebp), a glucose sensor in normal and cancer cells, interacted with AR in LNCaP cells. The interaction was further confirmed by coimmunoprecipitation analysis. Besides, Chrebp is required for the optimal transcriptional activity of AR in promoting the transcription of the prostate-specific antigen (PSA) promoter and messenger RNA (mRNA) expression. Consistently, knockdown of Chrebp using small interfering RNA (siRNA) in LNCaP cells reduced endogenous PSA levels. Together, our study demonstrates that Chrebp interacts with AR and regulates its transcriptional activity.

Prox1 Directly Interacts with LSD1 and Recruits the LSD1/NuRD Complex to Epigenetically Co-Repress CYP7A1 Transcription

Cholesterol 7α-hydroxylase (CYP7A1) catalyzes the first and rate-limiting step in the classical pathway of bile acids synthesis in liver and is crucial for maintaining lipid homeostasis. Hepatocyte nuclear factor 4α (HNF4α) and α1-fetoprotein transcription factor (FTF) are two major transcription factors driving CYP7A1 promoter activity in hepatocytes. Previous researches have shown that Prospero-related homeobox (Prox1) directly interacts with both HNF4α and FTF and potently co-represses CYP7A1 transcription and bile acid synthesis through unidentified mechanisms. In this work, mechanisms involved in Prox1-mediated co-repression were explored by identifying Prox1-associated proteins using immunoprecipitation followed by mass spectrometry (IP-MS) methodology. Multiple components of the epigenetically repressive lysine-specific demethylase 1 (LSD1)/nucleosome remodeling and histone deacetylase (NuRD) complex, most notably LSD1 and histone deacetylase 2 (HDAC2), were found to be associated with Prox1 and GST pulldown assay demonstrated that Prox1 directly interacts with LSD1. Sequential chromatin immunoprecipitation (ChIP) assays showed that Prox1 co-localizes with HNF4α, LSD1 and HDAC2 on CYP7A1 promoter in HepG2 cells. Furthermore, by using ChIP assay on HepG2 cells with endogenous Prox1 knocked down by RNA interference, Prox1 was shown to recruit LSD1 and HDAC2 onto CYP7A1 promoter and cause increased H3K4 demethylation. Finally, bile acids treatment of HepG2 cells, which significantly repressed CYP7A1 transcription, resulted in increased Prox1 and LSD1/NuRD complex occupancy on CYP7A1 promoter with a concurrent increase in H3K4 demethylation and H3/H4 deacetylation. These results showed that Prox1 interacts with LSD1 to recruit the repressive LSD1/NuRD complex to CYP7A1 promoter and co-represses transcription through epigenetic mechanisms. In addition, such Prox1-mediated epigenetic repression is involved in the physiologically essential negative feedback inhibition of CYP7A1 transcription by bile acids.

Amyloid-β metabolism in Niemann-Pick C disease models and patients

Niemann-Pick type C (NPC) is a progressive neurodegenerative lysosomal disease with altered cellular lipid trafficking. The metabolism of amyloid-β (Aβ) - previously mainly studied in Alzheimer's disease - has been suggested to be altered in NPC. Here we aimed to perform a detailed characterization of metabolic products from the amyloid precursor protein (APP) in NPC models and patients. We used multiple analytical technologies, including immunoassays and immunoprecipitation followed by mass spectrometry (IP-MS) to characterize Aβ peptides and soluble APP fragments (sAPP-α/β) in cell media from pharmacologically (U18666A) and genetically (NPC1 ( -/- ) ) induced NPC cell models, and cerebrospinal fluid (CSF) from NPC cats and human patients. The pattern of Aβ peptides and sAPP-α/β fragments in cell media was differently affected by NPC-phenotype induced by U18666A treatment and by NPC1 ( -/- ) genotype. U18666A treatment increased the secreted media levels of sAPP-α, AβX-40 and AβX-42 and reduced the levels of sAPP-β, Aβ1-40 and Aβ1-42, while IP-MS showed increased relative levels of Aβ5-38 and Aβ5-40 in response to treatment. NPC1 ( -/- ) cells had reduced media levels of sAPP-α and Aβ1-16, and increased levels of sAPP-β. NPC cats had altered CSF distribution of Aβ peptides compared with normal cats. Cats treated with the potential disease-modifying compound 2-hydroxypropyl-β-cyclodextrin had increased relative levels of short Aβ peptides including Aβ1-16 compared with untreated cats. NPC patients receiving β-cyclodextrin had reduced levels over time of CSF Aβ1-42, AβX-38, AβX-40, AβX-42 and sAPP-β, as well as reduced levels of the axonal damage markers tau and phosphorylated tau. We conclude that NPC models have altered Aβ metabolism, but with differences across experimental systems, suggesting that NPC1-loss of function, such as in NPC1 ( -/- ) cells, or NPC1-dysfunction, seen in NPC patients and cats as well as in U18666A-treated cells, may cause subtle but different effects on APP degradation pathways. The preliminary findings from NPC cats suggest that treatment with cyclodextrin may have an impact on APP processing pathways. CSF Aβ, sAPP and tau biomarkers were dynamically altered over time in human NPC patients.

Overlapping profiles of Abeta peptides in the Alzheimer's disease and pathological aging brains

IntroductionA hallmark of Alzheimer's disease (AD) is the presence of senile plaques composed of aggregated amyloid β (Aβ) peptides. Pathological aging (PA) is a postmortem classification that has been used to describe brains with plaque pathology similar in extent to AD, minimal cortical tau pathology, and no accompanying history of cognitive decline in the brain donor prior to death. PA may represent either a prodromal phase of AD, a benign form of Aβ accumulation, or inherent individual resistance to the toxic effects of Aβ accumulation. To attempt to distinguish between these possibilities we have systematically characterized Aβ peptides in a postmortem series of PA, AD and non-demented control (NDC) brains.MethodsAβ was sequentially extracted with tris buffered saline (TBS), radioimmunoprecipitation buffer (RIPA), 2% sodium dodecyl sulfate (SDS) and 70% formic acid (FA) from the pre-frontal cortex of 16 AD, eight PA, and six NDC patients. These extracts were analyzed by 1) a panel of Aβ sandwich ELISAs, 2) immunoprecipitation followed by mass spectrometry (IP/MS) and 3) western blotting. These studies enabled us to asses Aβ levels and solubility, peptide profiles and oligomeric assemblies.ResultsIn almost all extracts (TBS, RIPA, 2% SDS and 70% FA) the average levels of Aβ1-40, Aβ1-42, Aβ total, and Aβx-42 were greatest in AD. On average, levels were slightly lower in PA, and there was extensive overlap between Aβ levels in individual PA and AD cases. The profiles of Aβ peptides detected using IP/MS techniques also showed extensive similarity between the PA and AD brain extracts. In select AD brain extracts, we detected more amino-terminally truncated Aβ peptides compared to PA patients, but these peptides represented a minor portion of the Aβ observed. No consistent differences in the Aβ assemblies were observed by western blotting in the PA and AD groups.ConclusionsWe found extensive overlap with only subtle quantitative differences between Aβ levels, peptide profiles, solubility, and SDS-stable oligomeric assemblies in the PA and AD brains. These cross-sectional data indicate that Aβ accumulation in PA and AD is remarkably similar. Such data would be consistent with PA representing a prodromal stage of AD or a resistance to the toxic effects of Aβ.

Streamlined analysis schema for high-throughput identification of endogenous protein complexes

Immunoprecipitation followed by mass spectrometry (IP/MS) has recently emerged as a preferred method in the analysis of protein complex components and cellular protein networks. Targeting endogenous protein complexes of higher eukaryotes, particularly in large-scale efforts, has been challenging due to cellular heterogeneity, high proteome complexity, and, compared to lower organisms, lack of efficient in-locus epitope-tagging techniques. It is further complicated by variability in nonspecific identifications and cross-reactivity of primary antibodies. Still, the study of endogenous human protein networks is highly desired despite its challenges. Here we describe a streamlined IP/MS protocol for the purification and identification of extended endogenous protein complexes. We investigate the sources of nonspecific protein binding and develop semiquantitative specificity filters that are based on peptide spectral count measurements. We also outline logical constraints for the derivation of accurate complex composition from IP/MS data and demonstrate the effectiveness of this approach by presenting our analyses of different transcriptional coregulator complexes. We show consistent purification of novel components for the Integrator complex, analyze the composition of the Mediator complex solely from our data to demonstrate the wide usability of spectral counts, and deconvolute heterogeneous HDAC1/2 networks into core complex modules and several novel subcomplex interactions.