A Tandem-Affinity Purification Method for Identification of Primary Intracellular Drug-Binding Proteins.

In the field of drug discovery, understanding how small molecule drugs interact with cellular components is crucial. Our study introduces a novel methodology to uncover primary drug targets using Tandem Affinity Purification for identification of Drug-Binding Proteins (TAP-DBP). Central to our approach is the generation of a FLAG-hemagglutinin (HA)-tagged chimeric protein featuring the FKBP12(F36V) adaptor protein and the TurboID enzyme. Conjugation of drug molecules with the FKBP12(F36V) ligand allows for the coordinated recruitment of drug-binding partners effectively enabling in-cell TurboID-mediated biotinylation. By employing a tandem affinity purification protocol based on FLAG-immunoprecipitation and streptavidin pulldown, alongside mass spectrometry analysis, TAP-DBP allows for the precise identification of drug-primary binding partners. Overall, this study introduces a systematic, unbiased method for identification of drug-protein interactions, contributing a clear understanding of target engagement and drug selectivity to advance the mode of action of a drug in cells.

Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins.

Isolation of a protein/complex is important for its biochemical and structural characterization with mechanistic insights. TAP (tandem affinity purification) strategy allows rapid isolation of cellular proteins/complexes with a high level of purity. This methodology involves an immuno-affinity-based purification followed by a conformation-based isolation to obtain a highly homogeneous protein/complex. Here, we describe the TAP-mediated isolation of endogenous FACT (facilitates chromatin transcription; a heterodimer), an essential histone chaperone associated with BER (base excision repair). However, it is not clearly understood how FACT regulates BER. Such knowledge would advance our understanding of BER with implications in disease pathogenesis, since BER is an evolutionarily conserved process that is linked to various diseases including ageing, neurodegenerative disorders, and cancers. Using isolated FACT by TAP methodology, one can study the mechanisms of action of FACT in BER. Further, isolated FACT can be used for studies in other DNA transactions such as transcription and replication, as FACT is involved in these processes. Furthermore, TAP-mediated isolation strategy can be combined with mass spectrometry to identify the protein interaction partners of FACT.

Tandem Affinity Purification (TAP) of Interacting Prey Proteins with FLAG- and HA-Tagged Bait Proteins.

Proteins often interact with each other to form complexes and play functional roles in almost all cellular processes. The study of protein-protein interactions is therefore critical to understand protein function and biological pathways. Affinity Purification coupled with Mass Spectrometry (AP-MS) is an invaluable technique for identifying the interaction partners in protein complexes. In this approach, the protein of interest is fused to an affinity tag, followed by the expression and purification of the fusion protein. The affinity-purified sample is then analyzed by mass spectrometry to identify the interaction partners of the bait proteins. In this chapter, we detail the protocol for tandem affinity purification (TAP) based on the use of the FLAG (a fusion tag with peptide sequence DYKDDDDK) and hemagglutinin (HA) peptide epitopes. The immunoprecipitation using dual-affinity tags offers the advantage of increasing the specificity of the purification with lower nonspecific-background interactions.

Identification of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein partners using tandem affinity purification and mass spectrometry.

BACKGROUND: According to the World Health Organization Report, depressive disorders affect about 10% of the population. The molecular mechanism of the pathogenesis of depression is still not well understood. The new findings point to phosphatases as potential targets for effective depression therapy. The aim of the present work was the development of a method that would enable the identification of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein partners using a proteomic approach. METHODS: The research was carried out using the PC12 cell line, often used as a model for neurobiological research. The use of the procedure for efficient purification of protein complexes-tandem affinity purification (TAP) will facilitate the identification of proteins interacting with MKP-1, a potential goal of effective antidepressant therapy. RESULTS: Identified proteins belong to various groups: cytoskeletal, ribosomal, nucleic acid binding, chaperones, and enzymes and may potentially be involved in the molecular mechanism of depression. CONCLUSIONS: The presented protocol for the purification of protein complexes is universal and can be successfully used in different mammalian cell lines. Proteins identified in the present work have been reported in the literature concerning studies on depressive disorders, which speaks in favour of their role in depression.

Purification of hetero-oligomeric protein variants using a modified tandem affinity purification approach.

Here, we describe a protocol for artificially generating hetero-oligomeric protein complexes from the homo-oligomers using a sequential denaturation-renaturation strategy, followed by a modified affinity chromatography protocol used for their purification. This protocol enables one to obtain a homogenous population of hetero-oligomers and understand the contribution of each protomer through further biochemical and/or biophysical characterization. For complete details on the use and execution of this protocol, please refer to Parui et al. (2022).1.

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system.

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed "HiP4" (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein-protein interaction analyses.

Isolation of Protein Complexes from Tobacco Leaves by a Two-Step Tandem Affinity Purification.

Protein purification is an essential method for understanding protein function, as many biochemical and structural techniques require a high concentration of isolated protein for analysis. Yet, many studies of protein complexes are hampered by our inability to express them recombinantly in model systems, generally due to poor expression or aggregation. When studying a protein complex that requires its host cellular environment for proper expression and folding, endogenous purification is typically required. Depending on the protein of interest, however, endogenous purification can be challenging because of low expression levels in the host and lack of knowledge working with a non-model expression system, resulting in yields that are too low for subsequent analysis. Here, we describe a protocol for the purification of protein complexes endogenous to Nicotiana benthamiana directly from leaf tissue, with yields that enable structural and biochemical characterization. The protein complex is overexpressed in Nicotiana benthamiana leaves via agroinfiltration, and the protein-packed leaves are then mechanically ground to release the complex from the cells. The protein complex is finally purified by a simple two-step tandem affinity purification using distinct affinity tags for each complex member, to ensure purification of the assembled complex. Our method yields enough protein for various biochemical or structural studies. We have previously used this protocol to purify the complex formed by an innate immune receptor native to tobacco, ROQ1, and the Xanthomonas effector XopQ, and to solve its structure by single-particle cryo-electron microscopy-we use this example to illustrate the approach. This protocol may serve as a template for the purification of proteins from N. benthamiana that require the plant's cellular environment and are expressed at low levels. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression of the protein complex in leaf tissue Basic Protocol 2: Tandem affinity purification of the ROQ1-XopQ complex.

Protocol for establishing a protein-protein interaction network using tandem affinity purification followed by mass spectrometry in mammalian cells.

Identification of protein interactors is fundamental to understanding their functions. Here, we describe a modified protocol for tandem affinity purification coupled with mass spectrometry (TAP/MS), which includes two-step purification. We detail the S-, 2×FLAG-, and Streptavidin-Binding Peptide (SBP)- tandem tags (SFB-tag) system for protein purification. This protocol can be used to identify protein interactors and establish a high-confidence protein-protein interaction network based on computational models. This is particularly useful for identifying bona fide interacting proteins for subsequent functional studies. For complete details on the use and execution of this protocol, please refer to Bian et al. (2021).

Tandem affinity purification protocol for isolation of protein complexes from Schizosaccharomyces pombe.

Many cellular processes require the activities of complex molecular machines composed of several protein subunits. Insights into these systems can be gained by isolation of protein complexes followed by in vitro analyses determining the identity, posttranslational modifications, and interactions among proteins. Here, we present a protocol for tandem affinity purification (TAP) of protein complexes from the fission yeast Schizosaccharomyces pombe. The protocol employs cells expressing C-terminally TAP-tagged proteins and is suitable for the analysis of purified proteins by mass spectrometry. For complete information on the use and execution of this protocol, please refer to Cipakova et al. (2019).

Using Tandem Affinity Purification to Identify Circadian Clock Protein Complexes from Arabidopsis.

Identification of protein-protein interactions is an effective method of elucidating new roles for circadian clock-associated proteins that can expand beyond the information collected from transcriptional studies and genetic screens. Tandem affinity purification coupled with liquid chromatography mass spectrometry (APMS) utilizes epitope-tagged versions of your protein of interest to co-precipitate direct and indirect protein partners. Here, we provide a protocol and suggestions for proper design of 6x-His-3x-FLAG-tagged clock proteins and isolation of protein-protein interactions using two immunoprecipitation steps for increased specificity.

Expression and tandem affinity purification of 20S proteasomes and other multisubunit complexes in Haloferax volcanii.

Tandem affinity purification is a useful strategy to isolate multisubunit complexes of high yield and purity but can be limited when working with halophilic proteins that are not properly expressed in Escherichia coli. Halophilic proteins are desirable for bioindustrial applications as they are often stable and active in organic solvents; however, these proteins can be difficult to express, fold, and purify by traditional technologies. Haloarchaea provide a useful alternative for expression of halophilic proteins. These microorganisms use a salt-in strategy to maintain homeostasis and express most of their proteins with halophilic properties and low pI. Here, we provide detailed protocols for the genetic modification, expression and tandem affinity purification of "salt-loving" multisubunit complexes from the haloarchaeon Haloferax volcanii. The strategy for isolation of affinity tagged 20S proteasomes that form cylindrical proteolytic nanomachines of α1, α2 and ß subunits is described.

A protein interaction landscape of breast cancer.

Cancers have been associated with a diverse array of genomic alterations. To help mechanistically understand such alterations in breast-invasive carcinoma, we applied affinity purification­mass spectrometry to delineate comprehensive biophysical interaction networks for 40 frequently altered breast cancer (BC) proteins, with and without relevant mutations, across three human breast cell lines. These networks identify cancer-specific protein-protein interactions (PPIs), interconnected and enriched for common and rare cancer mutations, that are substantially rewired by the introduction of key BC mutations. Our analysis identified BPIFA1 and SCGB2A1 as PIK3CA-interacting proteins, which repress PI3K-AKT signaling, and uncovered USP28 and UBE2N as functionally relevant interactors of BRCA1. We also show that the protein phosphatase 1 regulatory subunit spinophilin interacts with and regulates dephosphorylation of BRCA1 to promote DNA double-strand break repair. Thus, PPI landscapes provide a powerful framework for mechanistically interpreting disease genomic data and can identify valuable therapeutic targets.

From Affinity to Proximity Techniques to Investigate Protein Complexes in Plants.

The study of protein-protein interactions (PPIs) is fundamental in understanding the unique role of proteins within cells and their contribution to complex biological systems. While the toolkit to study PPIs has grown immensely in mammalian and unicellular eukaryote systems over recent years, application of these techniques in plants remains under-utilized. Affinity purification coupled to mass spectrometry (AP-MS) and proximity labeling coupled to mass spectrometry (PL-MS) are two powerful techniques that have significantly enhanced our understanding of PPIs. Relying on the specific binding properties of a protein to an immobilized ligand, AP is a fast, sensitive and targeted approach used to detect interactions between bait (protein of interest) and prey (interacting partners) under near-physiological conditions. Similarly, PL, which utilizes the close proximity of proteins to identify potential interacting partners, has the ability to detect transient or hydrophobic interactions under native conditions. Combined, these techniques have the potential to reveal an unprecedented spatial and temporal protein interaction network that better understands biological processes relevant to many fields of interest. In this review, we summarize the advantages and disadvantages of two increasingly common PPI determination techniques: AP-MS and PL-MS and discuss their important application to plant systems.

Tandem Affinity Purification (TAP) of Low-Abundance Protein Complexes in Filamentous Fungi Demonstrated Using Magnaporthe oryzae.

Protein-protein interactions underlie cellular structure and function. In recent years, a number of methods have been developed for the identification of protein complexes and component proteins involved in the control of various biological pathways. Tandem affinity purification (TAP) coupled with mass spectrometry (MS) is a powerful method enabling the isolation of high-purity native protein complexes under mild conditions by performing two sequential purification steps using two different epitope tags. In this protocol, we describe a TAP-MS methodology for identifying protein-protein interactions present at very low levels in the fungal cell. Using the 6xHis-3xFLAG double tag, we start the affinity purification process for our protein of interest using high-capacity Ni2+ columns. This allows for greatly increased sample input compared to antibody-based first-step purification in conventional TAP protocols and provides a large amount of highly concentrated and preliminarily purified protein complexes to be used in a second purification step involving FLAG immunoprecipitation. The second step greatly facilitates the capture of low-level interacting partners under in vivo conditions. Our TAP-MS method has been proven to secure the characterization of low-abundance protein complexes under physiological conditions with high efficiency, specificity, and economy in the filamentous fungus Magnaporthe oryzae and might benefit gene function and proteomics studies in plants and other research fields.

A Highly Efficient Agrobacterium-Mediated Method for Transient Gene Expression and Functional Studies in Multiple Plant Species.

Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.

Protocol for Site-Specific Photo-Crosslinking Proteomics to Identify Protein-Protein Interactions in Mammalian Cells.

Protein-protein interactions (PPIs) play essential roles in almost all aspects of cellular processes. However, PPIs remain challenging to study due to their substoichiometry, low affinity, dynamic nature, and context dependence. Here, we present a protocol for the capture and identification of PPIs in live mammalian cells, which relies on site-specific photo-crosslinking in live cells, affinity purification, and quantitative proteomics. The protocol facilitates efficient and reliable identification of the interacting proteins of a given protein of interest in live cells. For complete details on the use and execution of this protocol, please refer to Wu et al. (2020).

Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins.

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly-acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging in Xenopus to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity- purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

Combined proximity labeling and affinity purification-mass spectrometry workflow for mapping and visualizing protein interaction networks.

Affinity purification coupled with mass spectrometry (AP-MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies. Whereas AP-MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP-MS and BioID data has been shown to comprehensively characterize a protein's molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP-MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform ( http://proteomics.fi ) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP-MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.

Genome-Wide Proteomics and Phosphoproteomics Analysis of Leishmania spp. During Differentiation.

Determining variations in protein abundance and/or posttranslational modification as a function of time or upon induction by a signal in a particular cell type is central to quantitative proteomics. Isobaric labeling methodologies now allow for parallel quantification of proteins at various conditions concurrently or multiplexing in relatively quantitative proteomics workflows. Hence, mapping the protein expression profiles of various developmental stages of Leishmania parasites is possible with high-resolution mass spectrometry. To analyze global changes in protein expression and cellular signaling pathways during Leishmania differentiation and development is possible with a quantitative proteomics approach. The tandem mass tags (TMT) approach provides a chemical labeling method based on the principle of amine reactive tags; the maximum number of conditions that can be multiplexed is 10-plex. We describe herein a detailed method for sample preparation, TMT-labeling, mass spectrometry and data analysis of different developmental stages of Leishmania donovani parasites. This quantitative proteomic approach is useful to study dynamic changes in protein expression levels during L. donovani differentiation, and also allows in-depth analysis of signaling pathways via phosphoproteomics.

Salivary Metabolomics of Total Body Irradiated Nonhuman Primates Reveals Long-Term Normal Tissue Responses to Radiation.

PURPOSE: To identify metabolomic biomarkers of acute radiation exposure in saliva that show time-dependent changes. METHODS AND MATERIALS: Nonhuman primates were exposed to 4 Gy of total body irradiation with γ-rays. Saliva was collected from 7 animals twice before and at days 1, 3, 5, 7, 15, 21, 28, and 60 after irradiation. Profiling was conducted with liquid chromatography time-of-flight mass spectrometry. Multivariate data analysis and potential biomarker identification was conducted through random Forests and the software MetaboAnalyst. Candidate biomarkers were validated through tandem mass spectrometry, and receiver operating characteristic curves were constructed to show the diagnostic ability of the signature over time. RESULTS: Untargeted metabolomic analysis revealed significant and persistent effects up to the 60 days evaluated in this study. Biomarkers spanning primarily amino acids and nucleotides were identified, with a significant number showing long-term responses. Fifteen biomarkers showed high statistical significance in the first week after irradiation and 16 at >7 days after irradiation (false discovery rate-adjusted P < .05). The combination of the biomarkers in a single biosignature was able to accurately show the diagnostic ability of the signature in a binary classifier system with receiver operating characteristic curves. CONCLUSIONS: Radiation can alter the metabolome in saliva, and metabolomics could effectively be used to monitor radiation responses, as a biodosimetry method, in the event of a radiological incident. Saliva metabolomics also has potential relevance in a clinical setting.