Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome

• Published in: Proteomes Vol. 5; no. 1; p. 3
• Main Authors: Kusch, Kathrin, Uecker, Marina, Liepold, Thomas, Möbius, Wiebke, Hoffmann, Christian, Neumann, Heinz, Werner, Hauke B, Jahn, Olaf
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 12.01.2017; MDPI
• Subjects: 2D gel electrophoresis (2DE) blot; Acetylation; acetylome; Antigens; Axons; cryo immuno-electron microscopy (IEM); cyclic nucleotide phosphodiesterase (CNP); Fluorescence; Gel electrophoresis; Gels; Host-pathogen interactions; immunoblot; Immunoblotting; immunoproteomics; isoelectric focusing (IEF); Lysine; Myelin; Post-translation; post-translational modification (PTM); Proteins; septin 8 (SEPT8); SERPA; Therapeutic applications; tubulin acetylation; myelin; post-translational modification (PTM); 2D gel electrophoresis (2DE) blot; immunoproteomics; SERPA; tubulin acetylation; cryo immuno-electron microscopy (IEM); acetylome; immunoblot; isoelectric focusing (IEF); septin 8 (SEPT8); cyclic nucleotide phosphodiesterase (CNP)
• ISSN: 2227-7382; 2227-7382
• DOI: 10.3390/proteomes5010003

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.