title: GOFAST: an integrated approach for efficient and comprehensive membrane proteome analysis.
abstract: Membrane proteomics, the large-scale analysis of membrane proteins, is often constrained by the difficulties of achieving fully resolvable separation and resistance to proteolysis, both of which could lead to low recovery and low identification rates of membrane proteins. Here, we introduce a novel integrated approach, GELFrEE Optimized FASP Technology (GOFAST) for large-scale and comprehensive membrane proteins analysis. Using an array of sample preparation techniques including gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), filter-aided sample preparation (FASP), and microwave-assisted on-filter enzymatic digestion, we identified 2 090 proteins from the membrane fraction of a leukemia cell line (K562). Of these, 37% are annotated as membrane proteins according to gene ontology analysis, resulting in the largest membrane proteome of leukemia cells reported to date. Our approach combines the advantages of GELFrEE high-loading capacity, gel-free separation, efficient depletion of detergents, and microwave-assisted on-filter digestion, minimizing sample losses and maximizing MS-detectable sequence coverage of individual proteins. In addition, this approach also shows great potential for the identification of alternative splicing products.
authors.affiliation: Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, North Carolina 27599, United States.
authors.forename: Yanbao
authors.initials: Y
authors.lastname: Yu
authors.forename: Ling
authors.initials: L
authors.lastname: Xie
authors.forename: Harsha P
authors.initials: HP
authors.lastname: Gunawardena
authors.forename: Jainab
authors.initials: J
authors.lastname: Khatun
authors.forename: Christopher
authors.initials: C
authors.lastname: Maier
authors.forename: Wendy
authors.initials: W
authors.lastname: Spitzer
authors.forename: Maarten
authors.initials: M
authors.lastname: Leerkes
authors.forename: Morgan C
authors.initials: MC
authors.lastname: Giddings
authors.forename: Xian
authors.initials: X
authors.lastname: Chen
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000379:methods*; D004586:Electrophoresis / Q000379:methods*; D005374:Filtration; D006801:Humans; D020014:K562 Cells; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*
chemical_list: D008565:Membrane Proteins; D020033:Protein Isoforms; D020543:Proteome