Alpha-synuclein is the major constituent of the Lewy bodies and Lewy neuritis which are the pathological hallmark of Parkinson's disease (PD). Quantitative assessment of the various alpha-synuclein forms in PD brains has not been reported. We developed an immunoprecipitation-mass spectrometry (IP-MS) assay and employed top-down MS with an internal N15-labeled standard spiked into both PD and control brain lysates to quantify alpha-synuclein forms using m/z signal normalized to the internal standard.We have successfully coupled an anti-alpha-synuclein monoclonal antibody to magnetic beads for an IP-MS assay that recovers nearly all alpha-synuclein in the brain tissue. Next, we validated the test-retest reliability of the IP-MS assay by establishing the assay variance. This was done by spiking in N15-labeled alpha-synuclein into 11 different lysates from the same brain on two separate days, performing IPs in duplicates followed by LC-MS injections for each immunoprecipitated sample. Raw m/z data from the most abundant charge state of each form of alpha-synuclein was used for quantitation in conjunction with 120 ng of N15-labeled alpha-synuclein spiked into each IP for normalization. Overall, we observed only 7.7% variance for the IP-MS assay from 22 LC-MS injections. We also established that the overall recovery of N15-labeled alpha-synuclein standard taken through the entire IP-MS analysis platform was about 70%. These data give us the confidence that we will be able to derive absolute concentrations (�g/mg) and identification of alpha-synuclein forms from PD and control brain tissues to achieve the objective of the study.An LTQ Orbitrap Velos is used in the study to achieve quantitative analysis and full protein characterization. In only two MS/MS scan events from a single charge state we identified fragment ions from endogenous alpha-synuclein from 60 of the possible 139 backbone cleavage sites spanning residues 4 to 137, thus demonstrating excellent characterization capability.

Quantitative assessment of alpha-synuclein forms in Parkinson's disease and normal brains by use of top-down mass spectrometry

246th National Meeting (2013)

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