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Characterization of α-Synuclein Multimer Stoichiometry in Complex Biological Samples by Electrophoresis.

ABSTRACT: The aberrant aggregation of α-synuclein in the brain is a hallmark of Parkinson's disease (PD). In vivo soluble α-synuclein occurs as a monomer and several multimers, the latter of which may be important for the biological function of α-synuclein. Currently, there is a lack of reproducible methods to compare α-synuclein multimer abundance between complex biological samples. Here we developed a method, termed "multimer-PAGE," that combines in-gel chemical cross-linking with several common electrophoretic techniques to measure the stoichiometry of soluble α-synuclein multimers in brain tissue lysates. Results show that soluble α-synuclein from the rat brain exists as several high molecular weight species of approximately 56 kDa (αS56), 80 kDa (αS80), and 100 kDa (αS100) that comigrate with endogenous lipids, detergents, and/or micelles during blue native gel electrophoresis (BN-PAGE). Co-extraction of endogenous lipids with α-synuclein was essential for the detection of soluble α-synuclein multimers. Homogenization of brain tissue in small buffer volumes (>50 mg tissue per 1 mL buffer) increased relative lipid extraction and subsequently resulted in abundant soluble multimer detection via multimer-PAGE. α-Synuclein multimers captured by directly cross-linking soluble lysates resembled those observed following multimer-PAGE. The ratio of multimer (αS80) to monomer (αS17) increased linearly with protein input into multimer-PAGE, suggesting to some extent, multimers were also formed during electrophoresis. Overall, soluble α-synuclein maintains lipid interactions following tissue disruption and readily forms multimers when this lipid-protein complex is preserved. Once the multimer-PAGE technique was validated, relative stoichiometric comparisons could be conducted simultaneously between 14 biological samples. Multimer-PAGE provides a simple inexpensive biochemical technique to study the molecular factors influencing α-synuclein multimerization.

SUBMITTER: Killinger BA

PROVIDER: S-EPMC4898865 | biostudies-other | 2016 Apr

REPOSITORIES: biostudies-other

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Project description:α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

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Characterization of α-Synuclein Multimer Stoichiometry in Complex Biological Samples by Electrophoresis.

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