ArticleAnalytical chemistry2010
Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes.
Article in Analytical chemistry, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Capillary Electrophoresis-Mass Spectrometry for Top-Down Proteomics.Annual review of analytical chemistry (Palo Alto, Calif.) · 2025Review
- Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles.Analytical chemistry · 2022Article
- Standard procedures for native CZE-MS of proteins and protein complexes up to 800 kDa.Electrophoresis · 2021Article
- Separation and Characterization of Endogenous Nucleosomes by Native Capillary Zone Electrophoresis-Top-Down Mass Spectrometry.Analytical chemistry · 2021Article
- Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization ofAnalytical chemistry · 2020Article
- Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry.Journal of the American Society for Mass Spectrometry · 2017Article
- Characterization of protein complexes and subcomplexes in protein-protein interaction databases.Biochemistry research international · 2015Article
- In-line separation by capillary electrophoresis prior to analysis by top-down mass spectrometry enables sensitive characterization of protein complexes.Journal of proteome research · 2014Article
- Protein analysis by shotgun/bottom-up proteomics.Chemical reviews · 2013Review
- Proteomics-based methods for discovery, quantification, and validation of protein-protein interactions.Analytical chemistry · 2013Review
- 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.Nature protocols · 2013Article
- Stable isotope labeling with amino acids in cell culture based mass spectrometry approach to detect transient protein interactions using substrate trapping.Analytical chemistry · 2011Article
- Mass Spectrometry-Based Proteomics Technologies to Define Endogenous Protein-Protein Interactions and Their Applications to Cancer and Viral Infectious Diseases.Mass spectrometry reviewsReview
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7 authors.
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Abstract
Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.
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