Automated Immunoprecipitation Workflow for Comprehensive Acetylome Analysis.

Immunoprecipitation is one of the most effective methods for enrichment of lysine-acetylated peptides for comprehensive acetylome analysis using mass spectrometry. Manual acetyl peptide enrichment method using non-conjugated antibodies and agarose beads has been developed and applied in various studies. However, it is time-consuming and can introduce contaminants and variability that leads to potential sample loss and decreased sensitivity and robustness of the analysis. Here we describe a fast, automated enrichment protocol that enables reproducible and comprehensive acetylome analysis using a magnetic bead-based immunoprecipitation reagent.

Analysis of N6-Methyladenosine Modification of HBV RNA by Methylated RNA Immunoprecipitation.

Of all the chemical modifications of RNAs, the N6-methyladenosine (m6A) modification is the most prevalent and well-characterized RNA modification that is functionally implicated in a wide range of biological processes. The m6A modification occurs in hepatitis B virus (HBV) RNAs and this modification regulates the HBV life cycle in several ways. Thus, understanding the mechanisms underlying m6A modification of HBV RNAs is crucial in understanding HBV infectious process and associated pathogenesis. Here, we describe the currently utilized method in the detection and characterization of m6A-methylated RNAs during viral infection.

Characterization of RVFV Nucleocapsid Protein Binding Sites on RNA by iCLIP-seq.

The nucleocapsid protein (N) in Rift Valley fever virus is an RNA-binding protein that functions in viral transcription, replication, and packaging. In this chapter, the method for studying protein-RNA interactions in context of viral infection using individual nucleotide resolution, cross-linking, immunoprecipitation, and sequencing (iCLIP-seq) is explained. The method is useful for identifying the interactions between both host and viral RNAs with N and can identify RNA motifs that interact with the protein of interest.

Nucleotide Resolution Mapping of Rift Valley Fever Virus Nucleoprotein-Genome RNA Interactions.

Rift Valley fever virus (RVFV; genus Phlebovirus, family Phenuiviridae, order Bunyavirales) is a mosquito-borne zoonotic pathogen endemic in Africa. Its negative-stranded genomic RNA (vRNA) is divided into three segments termed L, M, and S. Both vRNAs and antigenomic cRNAs are encapsidated by viral nucleoprotein (N) to form nucleocapsids, which constitute the template for genome transcription and replication. Based on a number of electron microscopy and structural studies, the viral RNAs of negative-strand RNA viruses, including phleboviruses, are commonly considered to be entirely and uniformly covered by N protein. However, high resolution data supporting this notion was missing to date.Here, we describe a method how to globally map all N-RNA interactions of RVFV by using iCLIP (individual-nucleotide resolution UV cross-linking and immunoprecipitation). The protocol is based on covalent cross-linking of direct protein-RNA interactions by UV irradiation. Following sample lysis, a selective isolation of N in complex with its RNA targets is achieved by immunoprecipitation. Then, N-RNA complexes are separated by SDS-PAGE, and after membrane transfer, RNA is isolated and subjected to library preparation and high-throughput sequencing. We explain how the standard iCLIP protocol can be adapted to RVFV N-RNA interaction studies. The protocol describes mapping of all N interactions with the vRNAs and cRNAs derived either from RVFV particles or from infected cells.

Mudskipper detects combinatorial RNA binding protein interactions in multiplexed CLIP data.

The uncovering of protein-RNA interactions enables a deeper understanding of RNA processing. Recent multiplexed crosslinking and immunoprecipitation (CLIP) technologies such as antibody-barcoded eCLIP (ABC) dramatically increase the throughput of mapping RNA binding protein (RBP) binding sites. However, multiplex CLIP datasets are multivariate, and each RBP suffers non-uniform signal-to-noise ratio. To address this, we developed Mudskipper, a versatile computational suite comprising two components: a Dirichlet multinomial mixture model to account for the multivariate nature of ABC datasets and a softmasking approach that identifies and removes non-specific protein-RNA interactions in RBPs with low signal-to-noise ratio. Mudskipper demonstrates superior precision and recall over existing tools on multiplex datasets and supports analysis of repetitive elements and small non-coding RNAs. Our findings unravel splicing outcomes and variant-associated disruptions, enabling higher-throughput investigations into diseases and regulation mediated by RBPs.

Immunoaffinity Intact-Mass Spectrometry for the Detection of Endogenous Concentrations of the Acetylated Protein Tumor Biomarker Neuron Specific Enolase.

Intact-mass spectrometry has huge potential for clinical application, as it enables both quantitative and qualitative analysis of intact proteins and possibly unlocks additional pathophysiological information via, e.g., detection of specific post-translational modifications (PTMs). Such valuable and clinically useful selectivity is typically lost during conventional bottom-up mass spectrometry. We demonstrate an innovative immunoprecipitation protein enrichment assay coupled to ultrahigh performance liquid chromatography quadrupole time-of-flight high resolution mass spectrometry (UPLC-QToF-HRMS) for the fast and simple identification of the protein tumor marker Neuron Specific Enolase Gamma (NSEγ) at low endogenous concentrations in human serum. Additionally, using the combination of immunoaffinity purification with intact mass spectrometry, the presence of NSEγ in an acetylated form in human serum was detected. This highlights the unique potential of immunoaffinity intact mass spectrometry in clinical diagnostics.

Extraction method combining saponin and trehalose useful for analyzing fragile intermolecular association.

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.

Unexpected discovery of CRP analytical interference: A case report.

OBJECTIVES: Immunoturbidimetric assays are sensitive techniques in clinical biology that may be subjected to matrix effects, hook effects or aspecific reactions. Among these, large quantities of immunoglobulins can distort the intensity of the detected signal. This study illustrates the deleterious effect of analytical interference on clinical patient management, and assesses the practical relevance of a recently proposed algorithm for interference investigation. METHODS: Determination of C-Reactive Protein (CRP) concentration by liquid immunoprecipitation on latex particles coated with mouse anti-CRP monoclonal antibodies, rabbit anti-CRP polyclonal antibodies, by solid phase immunochemistry or by enzymatic assay. RESULTS: During the follow-up of a 75-year-old patient suffering from multiple chronic diseases in the Internal Medicine Department of Toulouse University Hospital, a severe infection was suspected facing a CRP plasma value over 700 mg/L while he was in remission of an indolent marginal zone lymphoma. Because of the absence of clinical signs of infection, an interference in the liquid immunoprecipitation CRP assay was suspected. The hypothesis of an interference due to anti-mouse autoantibodies was ruled out because of normal results for other immunoassays using different types of antibodies. Moreover, no interference was observed using solid phase immunochemistry assay. Protein electrophoresis and immunofixation documented a relapse of lymphoma along with the presence of abnormal monoclonal immunoglobulins interfering with CRP measurement. CONCLUSION: The interpretation of common clinical biochemistry parameters such as CRP can be difficult owing to analytical interferences. Reviewing all the pharmaco-clinico-biological data and collaboration with clinicians is of critical importance for optimal patient management.

Protocol for qualitative analysis of lysosome immunoprecipitation from patient-derived glioblastoma stem-like cells.

Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting. For complete details on the use and execution of this protocol, please refer to Maghe et al.1.

MeRIP-Seq for Identifying Stress-Responsive Transcriptome-Wide m6A Profiles in Plants.

Recent advancements in detection and mapping methods have enabled researchers to uncover the biological importance of RNA chemical modifications, which play a vital role in post-transcriptional gene regulation. Although numerous types of RNA modifications have been identified in higher eukaryotes, only a few have been extensively studied for their biological functions. Of these, N6-methyladenosine (m6A) is the most prevalent and important mRNA modification that influences various aspects of RNA metabolism, including mRNA stability, degradation, splicing, alternative polyadenylation, export, and localization, as well as translation. Thus, they have implications for a variety of biological processes, including growth, development, and stress responses. The m6A deposition or removal on transcripts is dynamic and is altered in response to internal and external cues. Because this mark can alter gene expression under stress conditions, it is essential to identify the transcripts that can acquire or lose this epitranscriptomic mark upon exposure to stress conditions. Here we describe a step-by-step protocol for identifying stress-responsive transcriptome-wide m6A changes using RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq).

Isolation and Visualization of Plant Stress Granule-Associated Components via On-Beads Digestion and Co-localization Analysis.

Stress granules (SGs) are conserved cytoplasmic biomolecular condensates mainly formed by proteins and RNA molecules assembled by liquid-liquid phase separation. Isolation of SGs components has been a major challenge in the field due to the dynamic and transient nature of stress granule shells. Here, we describe the methodology for the isolation and visualization of SGs proteins from Arabidopsis thaliana plants using a scaffold component as the target. The protocol consists of the first immunoprecipitation of GFP-tagged scaffold protein, followed by an on-beads enzymatic digestion and previous mass spectrometry identification. Finally, the localization of selected SGs candidates is visualized in Nicotiana benthamiana mesophyll protoplasts.

Protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step immunoprecipitation approach.

Co-immunoprecipitation (coIP) is an experimental technique to study protein-protein interactions (PPIs). However, single-step coIP can only be used to identify the interaction between two proteins and does not solve the interaction testing of ternary complexes. Here, we present a protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step coIP approach. We describe steps for cell culture and transfection, elution of target proteins, and two-step coIP including western blot analyses. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Method for the Enrichment of N6-Methyladenosine-Modified Cellular and HIV-1 RNA.

N6-methyladenosine (m6A) modification of RNA is an important area in studying viral replication, cellular responses, and host immunity. HIV-1 RNA contains multiple m6A modifications that regulate viral replication and gene expression. HIV-1 infection of CD4+ T-cells or HIV-1 envelope protein treatment upregulates m6A levels of cellular RNA. Changes in the m6A modification of cellular transcripts in response to HIV-1 infection provide new insights into the mechanisms of posttranscriptional gene regulation in the host cell. To better investigate the functions of m6A modification in HIV-1 infection and innate immune responses, it is helpful to standardize basic protocols. Here, we describe a method for the selective enrichment of m6A-modified RNA from HIV-1-infected primary CD4+ T-cells based on immunoprecipitation. The enriched RNA with m6A modifications can be used in a variety of downstream applications to determine the methylation status of viral or cellular RNA at resolution from transcript level down to single nucleotide.

Development of a Gaussia luciferase immunoprecipitation assay for detecting Schistosoma japonicum infection.

Timely and accurate diagnosis of Schistosoma infection is important to adopt effective strategies for schistosomiasis control. Previously, we demonstrated that Schistosoma japonicum can secret extracellular vesicles and their cargos may serve as a novel type of biomarkers for diagnosing schistosomiasis. Here, we developed a Gaussia luciferase immunoprecipitation assay combined with S. japonicum extracellular vesicle (SjEV) protein to evaluate its potential for diagnosing schistosomiasis. A saposin-like protein (SjSLP) identified from SjEVs was fused to the Gaussia luciferase as the diagnostic antigen. The developed method showed good capability for detecting S. japonicum infection in mice and human patients. We also observed that the method could detect Schistosoma infection in mice as early as 7 days of post-infection, which showed better sensitivity than that of indirect ELISA method. Overall, the developed method showed a good potential for detecting Schistosoma infection particularly for early stage, which may provide an alternative strategy for identify Schistosoma infection for disease control.

Protocol to process crosslinking and immunoprecipitation data into annotated binding sites.

Here, we present a protocol for using Skipper, a pipeline designed to process crosslinking and immunoprecipitation (CLIP) data into annotated binding sites. We describe steps for partitioning annotated transcript regions and fitting data to a beta-binomial model to call windows of enriched binding. From raw CLIP data, we detail how users can map reproducible RNA-binding sites to call enriched windows and perform downstream analysis. This protocol supports optional customizations for different use cases. For complete details on the use and execution of this protocol, please refer to Boyle et al.1.

Mapping RNA-protein interactions with subcellular resolution using colocalization CLIP.

RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

Degradation-Based Protein Profiling: A Case Study of Celastrol.

Natural products, while valuable for drug discovery, encounter limitations like uncertainty in targets and toxicity. As an important active ingredient in traditional Chinese medicine, celastrol exhibits a wide range of biological activities, yet its mechanism remains unclear. In this study, they introduced an innovative "Degradation-based protein profiling (DBPP)" strategy, which combined PROteolysis TArgeting Chimeras (PROTAC) technology with quantitative proteomics and Immunoprecipitation-Mass Spectrometry (IP-MS) techniques, to identify multiple targets of natural products using a toolbox of degraders. Taking celastrol as an example, they successfully identified its known targets, including inhibitor of nuclear factor kappa B kinase subunit beta (IKKß), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3Kα), and cellular inhibitor of PP2A (CIP2A), as well as potential new targets such as checkpoint kinase 1 (CHK1), O-GlcNAcase (OGA), and DNA excision repair protein ERCC-6-like (ERCC6L). Furthermore, the first glycosidase degrader is developed in this work. Finally, by employing a mixed PROTAC toolbox in quantitative proteomics, they also achieved multi-target identification of celastrol, significantly reducing costs while improving efficiency. Taken together, they believe that the DBPP strategy can complement existing target identification strategies, thereby facilitating the rapid advancement of the pharmaceutical field.

Analysis of Protein Interactions in Patient-Derived Xenografts Using Immunoprecipitation.

Proteins are large, complex molecules that regulate multiple functions within the cell. The protein rarely functions as a single molecule, but rather interacts with one or more other proteins forming a dynamic network. Protein-protein interactions are critical for regulating the cell's response toward various stimuli from outside and inside the cell. Identification of protein-protein interactions enhanced our understanding of various biological processes in the living cell. Immunoprecipitation (IP) has been one of the standard and most commonly used biochemical methods to identify and confirm protein-protein interactions. IP uses a target protein-specific antibody conjugated with protein A/G affinity beads to identify molecules interacting with the target protein. Here, we describe the principle, procedure and challenges of the IP assay.

Evaluation of In Vivo Prepared Albumin-Drug Conjugate Using Immunoprecipitation Linked LC-MS Assay and Its Application to Mouse Pharmacokinetic Study.

There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38.

The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence.

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.