İnsan beyin dokusundan optimize edilmiş immünopresipitasyonu takiben yeni kesilmiş ⁇ -sinüklein türlerinin kütle spektrometrik tanımlaması.

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α-synuclein is a protein central to neurodegenerative diseases, and its functions are affected by multiple posttranslational modifications. Mass spectrometry is powerful for the characterization of α-synuclein forms but requires prior efficient immunoprecipitation conditions. In this study, we refined the immunoprecipitation of α-synuclein from human brain tissues by evaluating key parameters that influence recovery and specificity. We assessed the performance of tosyl-activated magnetic beads versus sheep antibody beads, identifying the optimal bead type for enhanced binding efficiency. Various elution conditions were rigorously tested to maximize protein yield. We also evaluated a range of antibodies specific to α-synuclein and delineated the effects of antibody amount and bead volume on the recovery of α-synuclein. The optimized immunoprecipitation protocol was effectively combined with high-resolution mass spectrometry for characterizing brain-derived α-synuclein from Parkinson's disease patients and controls. The assay identified a total of 38 N- or C-terminal truncated α-synuclein forms, including 22 novel sites. Our findings provide analytical tools for the reliable enrichment and characterization of α-synuclein from complex biological matrices, with potential applications in biomarker discovery and the investigation of pathogenic mechanisms underlying synucleinopathies.

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