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Published on: December 8, 2021
MS characterization of qualitative protein polymorphisms in the spinal cords of inbred mouse strains
Stefan Mikkat1, Peter Lorenz, Christian Scharf
1Core Facility Proteome Analysis, Medical Faculty, University of Rostock, Rostock, Germany. stefan.mikkat@med.uni-rostock.de
Abstract:
The spinal cord proteomes of two inbred mouse strains with different susceptibility to experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, were investigated by 2-DE and MALDI-MS. A proteome map comprising 304 different protein species was established. Using 2-D fluorescence difference gel electrophoresis, a comparison of the mouse strains revealed 26 qualitatively polymorphic proteins with altered electrophoretic mobility. MS analyses and DNA sequencing were applied to characterize their structural differences and 14 single amino acid substitutions were identified. Moreover, analysis of selectively enriched phosphopeptides from the neurofilament heavy polypeptide of both mouse strains revealed a high degree of diversity in the phosphorylated C-terminal domains of this protein. The described approach is capable to structurally characterize qualitative protein polymorphisms, whereas their functional significance remains to be elucidated. For some proteins formerly associated with experimental autoimmune encephalomyelitis and/or multiple sclerosis structural polymorphisms are described here, which may be subjected to further investigations. In addition, this work should be of general interest for proteomic analysis of inbred strains, because it shows potentials and constraints in the use of 2-DE analysis and MALDI-MS to detect and characterize structural protein polymorphisms.
Insights
This study mapped spinal cord proteins in mice, finding 14 amino acid differences linked to experimental autoimmune encephalomyelitis (EAE) susceptibility. Further research is needed to understand the functional impact of these protein variations.
Area of Science:
- Proteomics
- Neuroscience
- Genetics
Background:
- Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS).
- Inbred mouse strains exhibit varying susceptibility to EAE.
- Understanding protein differences in the spinal cord may reveal disease mechanisms.
Purpose of the Study:
- To investigate spinal cord proteomes in two mouse strains with differing EAE susceptibility.
- To identify and structurally characterize protein polymorphisms between the strains.
- To assess the utility of 2-DE and MALDI-MS for detecting protein variations.
Main Methods:
- Two-dimensional gel electrophoresis (2-DE) to separate proteins.
- Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for protein identification.
- DNA sequencing to confirm structural protein differences.
- Enrichment and analysis of phosphopeptides.
Main Results:
- Established a proteome map of 304 protein species from mouse spinal cords.
- Identified 26 qualitatively polymorphic proteins with altered electrophoretic mobility.
- Characterized 14 single amino acid substitutions between the mouse strains.
- Revealed significant diversity in phosphorylated C-terminal domains of neurofilament heavy polypeptide.
Conclusions:
- Developed an approach to structurally characterize protein polymorphisms using 2-DE and MALDI-MS.
- Identified specific protein structural variations potentially relevant to EAE and MS.
- Highlighted the potential and limitations of 2-DE and MALDI-MS in proteomic analysis of inbred strains.
- The functional significance of identified polymorphisms requires further investigation.
