title: Improved surfaceome coverage with a label-free nonaffinity-purified workflow.
abstract: The proteins of the cellular plasma membrane (PM) perform important functions relating to homeostasis and intercellular communication. Due to its overall low cellular abundance, amphipathic character, and low membrane-to-cytoplasm ratio, the PM proteome has been challenging to isolate and characterize, and is poorly represented in standard LC-MS/MS analyses. In this study, we employ sucrose gradient ultracentrifugation for the enrichment of the PM proteome, without chemical labeling and affinity purification, together with GeLCMS and use subsequent bioinformatics tools to select proteins associated with the PM/cell surface, herein referred to as the surfaceome. Using this methodology, we identify over 1900 cell surface associated proteins in a human acute myeloid leukemia cell line. These surface proteins comprise almost 50% of all detected cellular proteins, a number that substantially exceeds the depth of coverage in previously published studies describing the leukemia surfaceome.
authors.affiliation: Division of Oncology, Center for Childhood Cancer Research, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
authors.forename: Tina
authors.initials: T
authors.lastname: Glisovic-Aplenc
authors.affiliation: Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
authors.forename: Saar
authors.initials: S
authors.lastname: Gill
authors.affiliation: Protein and Proteomics Core Facility, The Children's Hospital of Philadelphia Research Institute, Philadelphia, PA, USA.
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Spruce
authors.affiliation: Protein and Proteomics Core Facility, The Children's Hospital of Philadelphia Research Institute, Philadelphia, PA, USA.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Smith
authors.affiliation: Protein and Proteomics Core Facility, The Children's Hospital of Philadelphia Research Institute, Philadelphia, PA, USA.
authors.forename: Hossein
authors.initials: H
authors.lastname: Fazelinia
authors.affiliation: Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Shestova
authors.affiliation: Protein and Proteomics Core Facility, The Children's Hospital of Philadelphia Research Institute, Philadelphia, PA, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Ding
authors.affiliation: Division of Oncology, Center for Childhood Cancer Research, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Tasian
authors.affiliation: Division of Oncology, Center for Childhood Cancer Research, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Aplenc
authors.affiliation: Protein and Proteomics Core Facility, The Children's Hospital of Philadelphia Research Institute, Philadelphia, PA, USA.
authors.forename: Steven H
authors.initials: SH
authors.lastname: Seeholzer
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002853:Chromatography, Liquid; D019295:Computational Biology / Q000379:methods*; D006801:Humans; D007962:Leukocytes / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification*; D013395:Sucrose / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D008565:Membrane Proteins; D013395:Sucrose
keywords: GO term; Label-free; Leukemia; Plasma membrane; Surfaceome