title: Protein Mobility Shifts Contribute to Gel Electrophoresis Liquid Chromatography Analysis.
abstract: Profiling of cellular and subcellular proteomes by liquid chromatography with tandem mass spectrometry (MS) after fractionation by SDS-PAGE is referred to as GeLC (gel electrophoresis liquid chromatography)-MS. The GeLC approach decreases complexity within individual MS analyses by size fractionation with SDS-PAGE. SDS-PAGE is considered an excellent fractionation technique for intact proteins because of good resolution for proteins of all sizes, isoelectric points, and hydrophobicities. Additional information derived from the mobility of the intact proteins is available after an SDS-PAGE fractionation, but that information is usually not incorporated into the proteomic analysis. Any chemical or proteolytic modification of a protein that changes the mobility of that protein in the gel can be detected. The ability of SDS-PAGE to resolve proteins with chemical modifications has not been widely utilized within profiling experiments. In this work, we examined the ability of the GeLC-MS approach to help identify proteins that were modified after a small hairpin RNA-dependent knockdown in an experiment using stable isotope labeling by amino acids in cell culture-based quantitation.
authors.affiliation: 1 Institute of Environmental Health Sciences and 2 Carman and Ann Adam Department of Pediatrics, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Carruthers
authors.affiliation: 1 Institute of Environmental Health Sciences and 2 Carman and Ann Adam Department of Pediatrics, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: Graham C
authors.initials: GC
authors.lastname: Parker
authors.affiliation: 1 Institute of Environmental Health Sciences and 2 Carman and Ann Adam Department of Pediatrics, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: Theresa
authors.initials: T
authors.lastname: Gratsch
authors.affiliation: 1 Institute of Environmental Health Sciences and 2 Carman and Ann Adam Department of Pediatrics, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Caruso
authors.affiliation: 1 Institute of Environmental Health Sciences and 2 Carman and Ann Adam Department of Pediatrics, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Stemmer
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D055785:Gene Knockdown Techniques; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D034741:RNA, Small Interfering; C526472:Smn1 protein, mouse; D055533:Survival of Motor Neuron 1 Protein
keywords: GeLC-MS; SDS-PAGE; SILAC; mass spectrometry; phosphoproteomics