title: CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis.
abstract: Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University; Institute of Chemistry, Proteomics Unit, Federal University of Rio de Janeiro.
authors.forename: Rafael D
authors.initials: RD
authors.lastname: Melani
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University.
authors.forename: Henrique S
authors.initials: HS
authors.lastname: Seckler
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University.
authors.forename: Owen S
authors.initials: OS
authors.lastname: Skinner
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University; Department of Cell Biology, Brazilian Center for Protein Research, Laboratory of Biochemistry and Protein Chemistry, University of Brasilia.
authors.forename: Luis H F
authors.initials: LH
authors.lastname: Do Vale
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Catherman
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University.
authors.forename: Pierre C
authors.initials: PC
authors.lastname: Havugimana
authors.affiliation: Department of Cell Biology, Brazilian Center for Protein Research, Laboratory of Biochemistry and Protein Chemistry, University of Brasilia.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Valle de Sousa
authors.affiliation: Institute of Chemistry, Proteomics Unit, Federal University of Rio de Janeiro.
authors.forename: Gilberto B
authors.initials: GB
authors.lastname: Domont
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University; philip-compton@northwestern.edu.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Compton
mesh_terms: D000818:Animals; D005591:Chemical Fractionation / Q000379:methods*; D004586:Electrophoresis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry*; D009206:Myocardium / Q000737:chemistry
chemical_list: D046912:Multiprotein Complexes