title: Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome.
abstract: Post-translational modifications (PTMs) play a key role in regulating protein function, yet their identification is technically demanding. Here, we present a straightforward workflow to systematically identify post-translationally modified proteins based on two-dimensional gel electrophoresis. Upon colloidal Coomassie staining the proteins are partially transferred, and the investigated PTMs are immunodetected. This strategy allows tracking back the immunopositive antigens to the corresponding spots on the original gel, from which they are excised and mass spectrometrically identified. Candidate proteins are validated on the same membrane by immunodetection using a second fluorescence channel. We exemplify the power of partial immunoblotting with the identification of lysine-acetylated proteins in myelin, the oligodendroglial membrane that insulates neuronal axons. The excellent consistency of the detected fluorescence signals at all levels allows the differential comparison of PTMs across multiple conditions. Beyond PTM screening, our multi-level workflow can be readily adapted to clinical applications such as identifying auto-immune antigens or host-pathogen interactions.
authors.affiliation: Department of Neurogenetics, Max-Planck-Institute of Experimental Medicine, 37075 Göttingen, Germany. kusch@em.mpg.de.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Kusch
authors.affiliation: Proteomics Group, Max-Planck-Institute of Experimental Medicine, 37075 Göttingen, Germany. uecker@em.mpg.de.
authors.forename: Marina
authors.initials: M
authors.lastname: Uecker
authors.affiliation: Proteomics Group, Max-Planck-Institute of Experimental Medicine, 37075 Göttingen, Germany. liepold@em.mpg.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Liepold
authors.affiliation: Department of Neurogenetics, Max-Planck-Institute of Experimental Medicine, 37075 Göttingen, Germany. moebius@em.mpg.de.|Center for Nanoscale Microscopy and Molecular Physiology of the Brain, 37075 Göttingen, Germany. moebius@em.mpg.de.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Möbius
authors.affiliation: Free Floater (Junior) Research Group Applied Synthetic Biology, Georg-August University Göttingen, Institute for Microbiology and Genetics, 37077 Göttingen, Germany. cho@accurion.com.
authors.forename: Christian
authors.initials: C
authors.lastname: Hoffmann
authors.affiliation: Free Floater (Junior) Research Group Applied Synthetic Biology, Georg-August University Göttingen, Institute for Microbiology and Genetics, 37077 Göttingen, Germany. heinz.neumann@mpi-dortmund.mpg.de.
authors.forename: Heinz
authors.initials: H
authors.lastname: Neumann
authors.affiliation: Department of Neurogenetics, Max-Planck-Institute of Experimental Medicine, 37075 Göttingen, Germany. hauke@em.mpg.de.
authors.forename: Hauke B
authors.initials: HB
authors.lastname: Werner
authors.affiliation: Proteomics Group, Max-Planck-Institute of Experimental Medicine, 37075 Göttingen, Germany. jahn@em.mpg.de.|Center for Nanoscale Microscopy and Molecular Physiology of the Brain, 37075 Göttingen, Germany. jahn@em.mpg.de.
authors.forename: Olaf
authors.initials: O
authors.lastname: Jahn
keywords: 2D gel electrophoresis (2DE) blot; SERPA; acetylome; cryo immuno-electron microscopy (IEM); cyclic nucleotide phosphodiesterase (CNP); immunoblot; immunoproteomics; isoelectric focusing (IEF); myelin; post-translational modification (PTM); septin 8 (SEPT8); tubulin acetylation