title: Native GELFrEE: a new separation technique for biomolecular assemblies.
abstract: The cadre of protein complexes in cells performs an array of functions necessary for life. Their varied structures are foundational to their ability to perform biological functions, lending great import to the elucidation of complex composition and dynamics. Native separation techniques that are operative on low sample amounts and provide high resolution are necessary to gain valuable data on endogenous complexes. Here, we detail and optimize the use of tube gel separations to produce samples proven compatible with native, multistage mass spectrometry (nMS/MS). We find that a continuous system (i.e., no stacking gel) with a gradient in its extent of cross-linking and use of the clear native buffer system performs well for both fractionation and native mass spectrometry of heart extracts and a fungal secretome. This integrated advance in separations and nMS/MS offers the prospect of untargeted proteomics at the next hierarchical level of protein organization in biology.
authors: Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, the Proteomics Center of Excellence, and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University , Evanston, Illinois 60208, United States.
authors: Owen S
authors: OS
authors: Skinner
authors: Luis H F
authors: LH
authors: Do Vale
authors: Adam D
authors: AD
authors: Catherman
authors: Pierre C
authors: PC
authors: Havugimana
authors: Marcelo Valle
authors: MV
authors: de Sousa
authors: Philip D
authors: PD
authors: Compton
authors: Neil L
authors: NL
authors: Kelleher
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005591:Chemical Fractionation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006321:Heart / Q000502:physiology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000032:analysis* / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine; D014242:Trichoderma / Q000254:growth & development / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D011506:Proteins