Identification of Mammalian Protein Complexes by Lentiviral-Based Affinity Purification and Mass Spectrometry

2011 ◽  
pp. 31-45 ◽  
Author(s):  
Zuyao Ni ◽  
Jonathan B. Olsen ◽  
Andrew Emili ◽  
Jack F. Greenblatt
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Mammalian Protein

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.

scholarly journals Tandem Affinity Purification and Mass Spectrometry (TAP‐MS) for the Analysis of Protein Complexes

2019 ◽  
Vol 96 (1) ◽  
Author(s):  
Guillaume Adelmant ◽  
Brijesh K. Garg ◽  
Maria Tavares ◽  
Joseph D. Card ◽  
Jarrod A. Marto
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification

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 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.

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