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Size-Based Proteome Fractionation through Polyacrylamide Gel Electrophoresis Combined with LC-FAIMS-MS for In-Depth Top-Down Proteomics.
Takemori, Ayako; Kaulich, Philipp T; Cassidy, Liam; Takemori, Nobuaki; Tholey, Andreas.
Afiliación
  • Takemori A; Advanced Research Support Center, Institute for Promotion of Science and Technology, Ehime University, Toon 790-8577, Ehime, Japan.
  • Kaulich PT; Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany.
  • Cassidy L; Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany.
  • Takemori N; Advanced Research Support Center, Institute for Promotion of Science and Technology, Ehime University, Toon 790-8577, Ehime, Japan.
  • Tholey A; Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany.
Anal Chem ; 94(37): 12815-12821, 2022 09 20.
Article en En | MEDLINE | ID: mdl-36069571
The combination of liquid chromatography (LC) and gas-phase separation by field-asymmetric ion mobility spectrometry (FAIMS) is a powerful proteoform separation system for top-down proteomics. Here, we present an in-depth top-down proteomics workflow, GeLC-FAIMS-MS, in which a molecular-weight-based proteome fractionation approach using SDS-polyacrylamide gel electrophoresis is performed prior to LC-FAIMS-MS. Since individual bands and their corresponding mass ranges require different compensating voltages (CVs), the MS parameters for each gel band and CV were optimized to increase the number and reliability of proteoform identifications further. We developed an easy-to-implement and inexpensive procedure combining the earlier established Passively Eluting Proteins from Polyacrylamide gels as Intact species (PEPPI) protocol with an optimized Anion-Exchange disk-assisted Sequential sample Preparation (AnExSP) method for the removal of stains and SDS. The protocol was compared with a methanol-chloroform-water (MCW)-based protein precipitation protocol. The results show that the PEPPI-AnExSP procedure is better suited for the identification of low-molecular-weight proteoforms, whereas the MCW-based protocol showed advantages for higher-molecular-weight proteoforms. Moreover, complementary results were observed with the two methods in terms of hydrophobicity and isoelectric points of the identified proteoforms. In total, 8500 proteoforms could be identified in a human proteome standard, showing the effectiveness of the gel-based sample fractionation approaches in combination with LC-FAIMS-MS.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteoma / Proteómica Tipo de estudio: Prognostic_studies Límite: Humans Idioma: En Revista: Anal Chem Año: 2022 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Estados Unidos
Texto completo
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Imprimir
XML
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteoma / Proteómica Tipo de estudio: Prognostic_studies Límite: Humans Idioma: En Revista: Anal Chem Año: 2022 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Estados Unidos