title: Online matrix removal platform for coupling gel-based separations to whole protein electrospray ionization mass spectrometry.
abstract: A fractionation method called gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) has been used to dramatically increase the number of proteins identified in top-down proteomic workflows; however, the technique involves the use of sodium dodecyl sulfate (SDS), a surfactant that interferes with electrospray ionization. Therefore, an efficient removal of SDS is absolutely required prior to mass analysis. Traditionally, methanol/chloroform precipitation and spin columns have been used, but they lack reproducibility and are difficult to automate. Therefore, we developed an in-line matrix removal platform to enable the direct analysis of samples containing SDS and salts. Only small molecules like SDS permeate a porous membrane and are removed in a manner similar to cross-flow filtration. With this device, near-complete removal of SDS is accomplished within 5 min and proteins are subsequently mobilized into a mass spectrometer. The new platform was optimized for the analysis of GELFrEE fractions enriched for histones extracted from human HeLa cells. All four core histones and their proteoforms were detected in a single spectrum by high-resolution mass spectrometry. The new method versus protein precipitation/resuspension showed 2- to 10-fold improved signal intensities, offering a clear path forward to improve proteome coverage and the efficiency of top-down proteomics.
authors.affiliation: Departments of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Ki Hun
authors.initials: KH
authors.lastname: Kim
authors.affiliation: Departments of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Compton
authors.affiliation: Departments of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: John C
authors.initials: JC
authors.lastname: Tran
authors.affiliation: Departments of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D002138:Calibration; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods; D006367:HeLa Cells; D006657:Histones / Q000302:isolation & purification*; D006801:Humans; D008567:Membranes, Artificial; D016062:Porosity; D020033:Protein Isoforms / Q000302:isolation & purification; D012015:Reference Standards; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D006657:Histones; D008567:Membranes, Artificial; D020033:Protein Isoforms; D012967:Sodium Dodecyl Sulfate
keywords: SDS cleanup; cross-flow filtration; gel-based separation; online matrix removal device; top-down proteomics
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