Characterization of In Vivo Protein Complexes via Chemical Cross-Linking and Mass Spectrometry

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.

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Three Dimensional Structures of Proteins and Protein Complexes from Chemical Cross-Linking and Mass Spectrometry: A Biochemical and Computational Overview

Current Proteomics ◽

10.2174/157016406777145694 ◽

2006 ◽

Vol 3 (1) ◽

pp. 1-21 ◽

Cited By ~ 9

Author(s):

Bulbul Chakravarti ◽

Steven Lewis ◽

Deb Chakravarti ◽

Alpan Raval

Keyword(s):

Mass Spectrometry ◽

Protein Complexes ◽

Three Dimensional ◽

Cross Linking ◽

Chemical Cross Linking

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8 Analysis of protein complexes using chemical cross-linking and mass spectrometry

Methods in Protein Biochemistry ◽

10.1515/9783110252361.127 ◽

2011 ◽

Author(s):

Alexander Leitner

Keyword(s):

Mass Spectrometry ◽

Protein Complexes ◽

Cross Linking ◽

Chemical Cross Linking

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

Methods in Molecular Biology - Clinical Proteomics ◽

10.1007/978-1-0716-1936-0_17 ◽

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

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Driving integrative structural modeling with serial capture affinity purification

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2007931117 ◽

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.

Download Full-text