Affiliations: [a] Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada
| [b] Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Canada
| [c] Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, Providence Health Care, Vancouver, Canada
Correspondence to: Dr. Mari DeMarco, St. Paul’s Hospital, 1081 Burrard St, Vancouver, Canada V6Z 1Y6. Tel.: +1
604 682 2344; E-mail: [email protected].
Abstract: Introduction:Improved analytical tools for detailed characterization of synucleins in pre-clinical models of Parkinson’s disease (PD) and related synucleinopathies are needed. Objective:Develop a multiple reaction monitoring (MRM) liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to quantify species-specific sequences and structural heterogeneity in soluble α- and β-synucleins in brain tissue. Methods:Using a proteolytic digestion workflow, the MRM LC-MS/MS method assayed six proteotypic peptides from the α-synuclein sequence; three unique to mouse or human α-synuclein and three conserved in α- and β-synuclein. For quantification, we used labeled α-synuclein as the internal standard and an external calibration curve. As proof of concept, the synuclein LC-MS/MS method was applied to brain tissue specimens from M83 transgenic PD mice, which overexpresses human α-synuclein, relative to wild-type littermate controls. Results:The synuclein MRM assay was linear over a wide concentration range (at least one order of magnitude). The assay had several advantages over ligand binding analytical methods, such as western blotting and enzyme-linked immunosorbent assays. These advantages included the ability to: quantify 1) total α-synuclein, 2) combined α- and β-synucleins, 3) species-specific contributions to total α-synuclein (e.g., in mice expressing both mouse and human α-synuclein), and 4) identify peptide-specific profile differences that may reflect post-translational modifications, all within a single analysis. Conclusion:With improved and expanded analytical characteristics coupled with a streamlined sample preparation workflow, the quantitative synuclein profiling LC-MS/MS assay provides a versatile and efficient platform to characterize synuclein biology in pre-clinical models and the potential for application to human tissues and fluids.
Keywords: Protein sequence, transgenic mice, mass spectrometry, tandem mass
spectrometry, Parkinson’s disease, synucleins