scholarly journals Driving Integrative Structural Modeling with Serial Capture Affinity Purification

2020 ◽  
Author(s):  
Xingyu Liu ◽  
Ying Zhang ◽  
Zhihui Wen ◽  
Yan Hao ◽  
Charles A.S. Banks ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Model ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Quantitative Imaging ◽  
Specific Protein ◽  
Cross Linking ◽  
Affinity Enrichment

AbstractStreamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here, we describe Serial Capture Affinity Purification (SCAP) where two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multi-step affinity enrichment of specific protein complexes. The multifunctional capabilities of these protein tagging systems also permit in vivo validation of interactions using FRET and FCCS quantitative imaging. When coupling SCAP to cross-linking mass spectrometry, an integrated structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC chromatin associated protein complex, culminating in a structural model with two SPINDOC docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3 Taken together, we present an integrated affinity purification, live cell imaging, and cross linking mass spectrometry approach for the building of integrative structural models of protein complexes.

scholarly journals Driving integrative structural modeling with serial capture affinity purification

2020 ◽  
Vol 117 (50) ◽  
pp. 31861-31870
Author(s):  
Xingyu Liu ◽  
Ying Zhang ◽  
Zhihui Wen ◽  
Yan Hao ◽  
Charles A. S. Banks ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Model ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Quantitative Imaging ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Cross Linking ◽  
Affinity Enrichment

Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.

Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes

2021 ◽  
pp. 217-232
Author(s):  
Lucía Quintana-Gallardo ◽  
Moisés Maestro-López ◽  
Jaime Martín-Benito ◽  
Miguel Marcilla ◽  
Daniel Rutz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Structural Characterization ◽  
Protein Complexes ◽  
Cross Linking

scholarly journals Getting more out of co-immunoprecipitation mass spectrometry experiments by reducing interference using FAIMS.

2021 ◽  
Author(s):  
Ching-Seng Ang ◽  
Joanna Sacharz ◽  
Michael G Leeming ◽  
Shuai Nie ◽  
Swati Varshney ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Modern Biology ◽  
Spectrometry Analysis ◽  
Affinity Enrichment ◽  
Unbiased Manner ◽  
Gas Phase Ion

Co-immunoprecipitation of proteins coupled to mass spectrometry has transformed modern biology understanding of protein interaction networks. These approaches exploit the selective isolation of tagged proteins by affinity enrichment / purification to identify protein binding partners at scale and in an unbiased manner. In instances where a suitable antibody is not be available it is common to graft synthetic tags such as FLAG or His Tags onto target protein sequences allowing the use of commercially available and validated antibodies for affinity purification. To allow the selective elution of protein complexes competitive displacement using a large molar excess of the tag peptide is widely used. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of a contaminating peptide. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device can be applied to FLAG-Tag and His-Tag pull down assay to increase the depth of protein coverage in these experiments. By excluding tag peptides based on their ion mobility profiles we demonstrate that single compensation voltage, or stepped compensation voltages strategies can significantly increase the coverage of total proteins by up to 2.5-fold and unique proteins by up to 15-fold versus experiments that do not use FAIMS. Combined these results highlight FAIMS is able to improve proteome depth by excluding interfering peptides without the need for additional sample handling or altering sample preparation protocols.

scholarly journals Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

2020 ◽  
Vol 117 (8) ◽  
pp. 4088-4098 ◽  
Author(s):  
Craig Gutierrez ◽  
Ilan E. Chemmama ◽  
Haibin Mao ◽  
Clinton Yu ◽  
Ignacia Echeverria ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Dynamics ◽  
Structural Changes ◽  
Structural Model ◽  
Protein Complexes ◽  
Cop9 Signalosome ◽  
Cross Linking ◽  
E3 Ligases ◽  
Protein Ubiquitination ◽  
Mass Spectometry

The COP9 signalosome (CSN) is an evolutionarily conserved eight-subunit (CSN1–8) protein complex that controls protein ubiquitination by deneddylating Cullin-RING E3 ligases (CRLs). The activation and function of CSN hinges on its structural dynamics, which has been challenging to decipher by conventional tools. Here, we have developed a multichemistry cross-linking mass spectrometry approach enabled by three mass spectometry-cleavable cross-linkers to generate highly reliable cross-link data. We applied this approach with integrative structure modeling to determine the interaction and structural dynamics of CSN with the recently discovered ninth subunit, CSN9, in solution. Our results determined the localization of CSN9 binding sites and revealed CSN9-dependent structural changes of CSN. Together with biochemical analysis, we propose a structural model in which CSN9 binding triggers CSN to adopt a configuration that facilitates CSN–CRL interactions, thereby augmenting CSN deneddylase activity. Our integrative structure analysis workflow can be generalized to define in-solution architectures of dynamic protein complexes that remain inaccessible to other approaches.

scholarly journals Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

2008 ◽  
Vol 183 (2) ◽  
pp. 223-239 ◽  
Author(s):  
Laura Trinkle-Mulcahy ◽  
Séverine Boulon ◽  
Yun Wah Lam ◽  
Roby Urcia ◽  
François-Michel Boisvert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Cell Biology ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Specific Protein ◽  
Protein Interaction Data ◽  
Quantitative Mass Spectrometry ◽  
Cell Extracts ◽  
Interaction Partners

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

scholarly journals Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass-spectrometry

2020 ◽  
Author(s):  
Johannes F. Hevler ◽  
Marie V. Lukassen ◽  
Alfredo Cabrera-Orefice ◽  
Susanne Arnold ◽  
Matti F. Pronker ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Model ◽  
Protein Complexes ◽  
Protein Structures ◽  
Substantial Reduction ◽  
Cross Linking ◽  
Complement Components ◽  
Native Page ◽  
Blue Native Page ◽  
Cross Links

AbstractCross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS observed by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of artificial over-length cross-links when compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can be used to provide new insights into the initial stages of the terminal complement pathway.

scholarly journals RNA Proximity Labeling: A New Detection Tool for RNA–Protein Interactions

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2270
Author(s):  
Ronja Weissinger ◽  
Lisa Heinold ◽  
Saira Akram ◽  
Ralf-Peter Jansen ◽  
Orit Hermesh
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Cellular Functions ◽  
Cellular Compartments ◽  
Rna Protein Complexes

Multiple cellular functions are controlled by the interaction of RNAs and proteins. Together with the RNAs they control, RNA interacting proteins form RNA protein complexes, which are considered to serve as the true regulatory units for post-transcriptional gene expression. To understand how RNAs are modified, transported, and regulated therefore requires specific knowledge of their interaction partners. To this end, multiple techniques have been developed to characterize the interaction between RNAs and proteins. In this review, we briefly summarize the common methods to study RNA–protein interaction including crosslinking and immunoprecipitation (CLIP), and aptamer- or antisense oligonucleotide-based RNA affinity purification. Following this, we focus on in vivo proximity labeling to study RNA–protein interactions. In proximity labeling, a labeling enzyme like ascorbate peroxidase or biotin ligase is targeted to specific RNAs, RNA-binding proteins, or even cellular compartments and uses biotin to label the proteins and RNAs in its vicinity. The tagged molecules are then enriched and analyzed by mass spectrometry or RNA-Seq. We highlight the latest studies that exemplify the strength of this approach for the characterization of RNA protein complexes and distribution of RNAs in vivo.

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