title: Isolation, Proteomic Analysis, and Microscopy Confirmation of the Liver Nuclear Envelope Proteome.
abstract: Nuclei can be relatively easily extracted from homogenized liver due to the softness of the tissue and crudely separated from other cellular organelles by low-speed centrifugation due to the comparatively large size of nuclei. However, further purification is complicated by nuclear envelope continuity with the endoplasmic reticulum, invaginations containing mitochondria, and connections to the cytoskeleton. Subsequent purification to nuclear envelopes is additionally confounded by connections of inner nuclear membrane proteins to chromatin. For these reasons, it is necessary to confirm proteomic identification of nuclear envelope proteins by testing targeting of individual proteins. The proteomic identification of nuclear envelope fractions is affected by the tendencies of transmembrane proteins to have extreme isoelectric points, strongly hydrophobic peptides, posttranslational modifications, and a propensity to aggregate, thus making proteolysis inefficient. To circumvent these problems, we have developed a MudPIT approach that uses multiple extractions and sequential proteolysis to increase identifications. Here we describe methods for isolating nuclear envelopes, determining their proteome by MudPIT, and confirming their targeting to the nuclear periphery by microscopy.
authors.affiliation: The Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, University of Edinburgh, Edinburgh, EH9 3BF, UK.
authors.forename: Nadia
authors.initials: N
authors.lastname: Korfali
authors.affiliation: The Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO, 64110, USA. LAF@stowers.org.
authors.forename: Laurence
authors.initials: L
authors.lastname: Florens
authors.affiliation: The Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, University of Edinburgh, Edinburgh, EH9 3BF, UK. e.schirmer@ed.ac.uk.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Schirmer
mesh_terms: D000818:Animals; D053801:Capillary Electrochromatography; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D019295:Computational Biology; D008099:Liver / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000378:metabolism*; D021381:Protein Transport; D020543:Proteome; D040901:Proteomics / Q000379:methods; D051381:Rats; D053719:Tandem Mass Spectrometry
chemical_list: D008565:Membrane Proteins; D020543:Proteome
keywords: Digitonin permeabilization; Fluorescence microscopy; Inner nuclear membrane; Multidimensional protein identification technology (MudPIT); Nuclear envelope; Nuclear lamina; Transmembrane protein