ArticleNature protocols2013
A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry.
Article in Nature protocols, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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16 citing papers in PubMed, 29 citations in OpenAlex.
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- LAMTOR/Ragulator is a negative regulator of Arl8b- and BORC-dependent late endosomal positioning.The Journal of cell biology · 2017Article
- Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein.PLoS pathogens · 2017Article
- A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor.Nature chemical biology · 2017Article
- Expanding the Interactome of the Noncanonical NF-κB Signaling Pathway.Journal of proteome research · 2016Article
- An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.Molecular & cellular proteomics : MCP · 2016Article
- Characterization of Two Distinct Nucleosome Remodeling and Deacetylase (NuRD) Complex Assemblies in Embryonic Stem Cells.Molecular & cellular proteomics : MCP · 2016Article
- Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2-MND1.Journal of proteome research · 2015Article
- Article
- JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia.Nature genetics · 2014Article
- abFASP-MS: affinity-based filter-aided sample preparation mass spectrometry for quantitative analysis of chemically labeled protein complexes.Journal of proteome research · 2014Article
- Deregulated expression of TANK in glioblastomas triggers pro-tumorigenic ERK1/2 and AKT signaling pathways.Oncogenesis · 2013Article
- Affinity purification strategies for proteomic analysis of transcription factor complexes.Journal of proteome research · 2013Article
- The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.Nature methods · 2013Article
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Authors and funding
7 authors at 4 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis. This branch of the methodology amalgamates standard and well-practiced laboratory methods to reveal compositional changes that occur in protein complex architecture. By using mouse N-terminally tagged streptavidin-binding peptide-hemagglutinin-TANK-binding kinase 1 (SH-TBK1), we chemically cross-linked the affinity-purified complex of SH-TBK1 with the homobifunctional lysine-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), and we separated the resultant protein complexes by denaturation and by silver-stained one- and two-dimensional SDS-PAGE. We observed a range of cross-linked TBK1 complexes of variable pI and M(r) and confirmed them by immunoblotting. LC-MS analysis of in situ-digested cross-linked proteins shows differences in the composition of the TBK1 subcomplexes. The protocol is inherently simple and can be readily extended to the investigation of a range of protein complexes. From cell lysis to data generation by LC-MS, the protocol takes approximately 2.5 to 5.5 d to perform.
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