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Fluorescence-based assays are extremely diverse, sensitive and robust experimental methods for investigating the conformational changes, enzyme kinetics, dynamics and molecular interactions. A prerequisite for most of these experimental approaches is to label the protein of interest with one or more extrinsic fluorophores with desired photophysical properties. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the bottlenecks in this labelling reaction is requirement of high protein concentration, maintenance of protein stability during the labelling process as well as high background fluorescence due to ineffective removal of unreacted FITC, prior to fluorescence studies. Therefore, to overcome these inadequacies or limitations, we have modified the existing protocol by introducing tandem affinity purification tags at the N- and C-terminus of target protein. Using this modified method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence of unreacted FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in spectroscopic studies.

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. This protocol describes the MAC-tag approach, which combines affinity purification and biotinylation identification proximity labeling in a single tag. Binding proteins are identified by liquid chromatography–mass spectrometry, followed by visualization of protein localization using an online platform.

De novo fatty acid synthesis in plants occurs primarily in the plastids and is catalysed by a type-II fatty acid synthase (FAS) in which separate enzymes catalyse sequential reactions. Genes encoding all of the plant FAS components have been identified, following enzyme purification or by homology to Escherichia coli genes, and the structure of a number of the individual proteins determined. There are several lines of biochemical evidence indicating that FAS enzymes form a multi-protein complex and both in vitro and in vivo strategies can be used to investigate the association and interactions between them. To investigate protein interactions in vivo, tandem affinity purification-tagged FAS components are being used to purify complexes from both Arabidopsis thaliana and Synechocystis PCC6803. Here, the development of the tandem affinity purification method, its modification, and its use in plants is described and the experimental results achieved so far are reported.

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization. This Protocol describes how to perform online buffer exchange (OBE) coupled to native mass spectrometry (nMS) for rapid screening of structural features of pre-purified proteins, protein complexes or clarified cell lysates.

Essentially all cellular processes are orchestrated by protein-protein interactions (PPIs). In recent years, affinity purification coupled to mass spectrometry (AP-MS) has been the preferred method to identify cellular PPIs. Here we present a microfluidic-based AP-MS workflow, called on-chip AP-MS, to identify PPIs using minute amounts of input material. By using this automated platform we purify the human Cohesin, CCC and Mediator complexes from as little as 4 micrograms of input lysate, representing a 50─100-fold downscaling compared to regular microcentrifuge tube-based protocols. We show that our platform can be used to affinity purify tagged baits as well as native cellular proteins and their interaction partners. As such, our method holds great promise for future biological and clinical AP-MS applications in which sample amounts are limited. Affinity purification-mass spectrometry (AP-MS) can identify endogenous protein interactions but the need for high amounts of input material still limits its applicability. Here, the authors present a microfluidic-based AP-MS workflow that can capture protein interactions from 50─100-fold less input material than conventional approaches.

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

Plant bZIP group I transcription factors have been reported mainly for their role during vascular development and osmosensory responses. Interestingly, bZIP29 has been identified in a cell cycle interactome, indicating additional functions of bZIP29 in plant development. Here, bZIP29 was functionally characterized to study its role during plant development. It is not present in vascular tissue but is specifically expressed in proliferative tissues. Genome-wide mapping of bZIP29 target genes confirmed its role in stress and osmosensory responses, but also identified specific binding to several core cell cycle genes and to genes involved in cell wall organization. bZIP29 protein complex analyses validated interaction with other bZIP group I members and provided insight into regulatory mechanisms acting on bZIP dimers. In agreement with bZIP29 expression in proliferative tissues and with its binding to promoters of cell cycle regulators, dominant-negative repression of bZIP29 altered the cell number in leaves and in the root meristem. A transcriptome analysis on the root meristem, however, indicated that bZIP29 might regulate cell number through control of cell wall organization. Finally, ectopic dominant-negative repression of bZIP29 and redundant factors led to a seedling-lethal phenotype, pointing to essential roles for bZIP group I factors early in plant development.

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K + channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K + channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease. Synopsis Systems biology has the potential to explain physiological processes as emergent properties of sets of genes and proteins. Beyond simple cellular systems, the challenge of delivering systems biology into the intact and freely behaving animal will require new methods. Currently, the most widely used approach is immunoprecipitation of the target protein and its associate binding proteins. This method suffers from the drawbacks of single step purification strategies that include a high level of non‐specific background proteins amongst other limitations. To overcome these limitations we demonstrate that the tandem affinity purification (TAP) technology originally developed in yeast (Rigaut et al , 1999 ), when combined with gene targeting, can be used to efficiently isolate highly specific complexes from mouse. The ‘targeted TAP tagging’ strategy combines the two major advantages of each system. The first advantage is that the insertion of two tags into the protein of interest allows two consecutive purification steps that facilitate the recovery of protein complexes with high confidence and decreases the recovery of non‐specific proteins or weak interactors. The second advantage, conferred by targeting the endogenous gene, is that the tagged protein is expressed under its natural regulatory mechanisms. We have designed an endogenous TAP targeting strategy to isolate complexes from mouse brain excitatory synapses. The brain is the most complex organ from a cellular and molecular perspective and thus an ideal model to explore the TAP method. Post Synaptic Density 95 (PSD‐95/Dlg4) is an adaptor protein comprised of PDZ, SH3 and GK domains and is expressed in the postsynaptic terminal of excitatory synapses where it organizes signaling from neurotransmitter receptors to downstream pathways (Kornau et al , 1995 ; Hunt et al , 1996 ; Tu et al , 1999 ; Husi et al , 2000 ; Nehring et al , 2000 ; Dosemeci et al , 2007 ; Carlisle et al , 2008 ). Mice carrying a knockout mutation in PSD‐95 show it is essential for synaptic plasticity and a range of important behaviours (Migaud et al , 1998 ; El‐Husseini et al , 2000 ; Beique et al , 2006 ). Here, a new TAP tag was fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function (Figure 2 ). As a result of four independent tandem purifications and mass spectrometry analysis, we were able to define PSD‐95 core complexes with high sensitivity and reproducibility. The four purifications show an average of 125±19 proteins, having 118 proteins (94%) common in at least three of four replicates. This reproducibility rate is among the highest rate reported for systematic protein complex isolation. To further validate this interaction data we compared it to information from public datasets. Of the 118 proteins, 22% were proteins that directly bind PSD‐95 and 18% were proteins not previously found in other PSD‐95 analysis. All together, these data show robust reproducibility and sensitivity of this method for purifying synaptic complexes. These PSD‐95 core complexes comprise key functional components of synapses including the glutamate neurotransmitter receptors, K + channels, scaffolding and signaling proteins. These complexes contain ionotropic glutamate receptors of the NMDA, AMPA and kainate subtypes as well as major K + channels that together are the major postsynaptic constituents responsible for synaptic transmission and shaping the postsynaptic electrophysiological response to presynaptic input (Watanabe et al , 2002 ; Chen et al , 2006 ; Kim et al , 2007 ). We believe that this is the first method that has allowed the robust copurification of these proteins. To explore functional organization using network models, we manually curated interactions (Pocklington et al , 2006 ) and the UniHi database ( http://www.mdc‐berlin.de/unihi ) to identify 119 interactions between 50 proteins (excluding self‐interactions) of the PSD‐95 core complexes. Network clustering of the interacting proteins showed 40 out of the 50 proteins formed a large connected component (major connected component, MCC) and a modular structure that was segregated into 5 clusters referred to as cluster a (Cla) to cluster e (Cle) (Figure 5A ). In addition to the 5 MCC clusters, 2 further disconnected clusters (‘Clf’ and ‘Clg’) were found. Of great interest is the location and proximity of the receptors and channels responsible for the postsynaptic depolarization and subsequent action potential generation. All NMDA, AMPA and kainate glutamate receptors were restricted to Cla and Clb and the voltage‐dependent K + channels were found in Cla and Clc (entirely comprised of K + channels). It therefore appears that Cla, Clb and Clc are enriched with membrane proteins responsible for electrical properties of the postsynaptic terminal. The central role of PSD‐95 was supported by calculation of the shortest path from each protein to every other protein and PSD‐95 showed the lowest. Annotation of clusters with human disease associations revealed that multiple disorders map onto the network with a highly significant correlation of schizophrenia within the glutamate receptor clusters ( P <10−6). 20 genes involved in schizophrenia were significantly associated with the clusters Cla and Clb that contains all the glutamate receptors and MAGUK/Dlg proteins (Figure 5B ). Mapping the primary interactors of these schizophrenia proteins recruited many other proteins found in the other modules of the network. This suggests that the overall network and its different modules are a substrate for schizophrenia, and not simply the glutamate receptors, as was generally considered in the ‘glutamate hypothesis’ of schizophrenia (Greene, 2001 ; Coyle, 2006 ; Lisman et al , 2008 ). This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease. TAP tagged mice are a valuable resource and useful for a wide range of physiological studies and whole animal studies. A novel approach for isolating native protein complexes from mouse tissues using gene targeting of tandem affinity tags is presented. A protein core complex from brain synapses comprising principal electrophysiological and signalling components for synaptic transmission and synaptic plasticity was isolated. The protein interaction network shows clusters of functionally distinct proteins and schizophrenia susceptibility genes. This targeted TAP tagging method has general application to all types of protein complexes in the mouse and will be particularly useful for analysing molecular networks and systems biology in the intact animal.

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.

Histone 3 lysine 4 methylation (H3K4me) is an evolutionarily conserved epigenetic marker associated with transcriptional activation, playing a crucial role in growth and development. In yeast, all forms of H3K4 methylation are catalyzed by the COMPASS complex. However, purifying endogenous COMPASS remains challenging due to its low abundance, compositional complexity, and structural instability, resulting in low yield, poor purity, and heterogeneity in isolated complexes. These technical limitations have impeded the structural elucidation of the intact COMPASS complex and contributed to inconsistencies in reported in vitro enzymatic activity, thereby limiting a comprehensive understanding of its functions. Here, we present an optimized tandem affinity purification strategy that enables the high-yield isolation of native COMPASS from Saccharomyces cerevisiae with >99% purity and intact subunit composition, as validated by biochemical analyses. Using recombinant nucleosomes as substrates, we systematically characterized its catalytic properties and found that endogenously purified COMPASS exhibited strict dependence on H2B ubiquitination for catalyzing H3K4 methylation. This work establishes an efficient purification strategy for future structural and functional studies of COMPASS and provides critical insights into its catalytic properties.