title: Statically Adsorbed Coatings for High Separation Efficiency and Resolution in CE-MS Peptide Analysis: Strategies and Implementation.
abstract: Coatings are necessary to prevent protein and peptide adsorption to the capillary surface and obtain high intermediate precision. In this protocol, we first present our basic strategy to address peptide separation using three different coatings: one neutral and two cationic coatings, the latter largely differing in their induced electroosmotic mobility. In detail, we will describe how we apply the statically adsorbed coatings to obtain very high plate numbers and high repeatability.With some model examples, we clearly describe the scope of the method for the analysis of peptide samples: tryptic digests are addressed as well as small glycoproteins and glycopeptides largely differing in their effective electrophoretic mobility. We also show that the method is suitable for a fast screening of peptide samples despite a high matrix load comprising of up to 500 mmol/L sodium chloride. We demonstrate that this basic CE-MS method is rather independent of the polarity of the analytes with a very fast near-baseline separation of very hydrophobic Aβ peptides related to the onset of Alzheimer's disease. These examples will give an impression, which coating is most suitable for a specific analytical application.Special attention is paid to difficult aspects of the coating procedure and the CE-MS method, e.g., the potential of cross-contamination when changing the coatings.
authors.affiliation: Institute for Physical and Theoretical Chemistry, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 18, Tübingen, 72076, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Pattky
authors.affiliation: Functional Proteomics, Medical Proteome-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Katalin
authors.initials: K
authors.lastname: Barkovits
authors.affiliation: Functional Proteomics, Medical Proteome-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
authors.affiliation: Institute of Complex Systems, ICS-6: Structural Biochemistry, Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Oliver H
authors.initials: OH
authors.lastname: Weiergräber
authors.affiliation: Institute for Physical and Theoretical Chemistry, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 18, Tübingen, 72076, Germany. carolin.huhn@uni-tuebingen.de.
authors.forename: Carolin
authors.initials: C
authors.lastname: Huhn
mesh_terms: D000327:Adsorption; D002412:Cations / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002412:Cations; D010455:Peptides; D011506:Proteins
keywords: Coatings; Electroosmotic flow velocity; Mass spectrometry; Peptides; Proteins; Resolution