Identification of Novel α-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

Abstract

Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated α-synuclein (α-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of α-syn in brain tissue homogenates. N-terminally acetylated full-length α-syn (Ac-α-syn1–140) and two N-terminally acetylated C-terminally truncated forms of α-syn (Ac-α-syn1–139 and Ac-α-syn1–103) were found. The different forms of α-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of α-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA).

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

“…Previous studies have suggested that native ␣-syn is subjected to N-terminal acetylation (35,36). Using tandem mass spectrometry of GluC-digested samples, we now confirm that RBC ␣-syn is quantitatively acetylated at its N terminus, thus establishing that this modification is ubiquitous and not restricted to ␣-syn in the brain (35,36).…”

“…To allow more accurate assessment and comparison of the oligomeric state of ␣-syn derived from different sources, the following protein standards of well-defined chemical integrity and purity (assessed by SDS-PAGE and mass spectrometry), as well as conformational properties (measured by CD) and oligomeric state (determined by size-exclusion chromatography coupled to light scattering) were generated and used as controls in all the studies presented below: 1) unfolded monomeric ␣-syn from E. coli; 2) disulfide-linked ␣-syn dimer (A140C mutation) produced in E. coli; and 3) ␣-syn specifically acetylated at the N terminus. Unlike ␣-syn produced in E. coli, the majority of ␣-syn expressed in the brain and in mammalian cell lines undergoes N-terminal acetylation (35,36). Direct comparison of these standards and native ␣-syn from different sources on the same gel is particularly important, because the migration profile of a given protein in native gel electrophoresis is dependent on its molecular weight, conformation and charge state, as well as the percentage of acrylamide within the gel.…”

Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach

“…In this study we used a hybrid approach that combined affinity purification through immunoprecipitation with a monoclonal antibody and quantitation and characterization with mass spectrometry analysis (22). Brain tissue from agematched patients with AD (n ϭ 15) and controls (n ϭ 15) (supplemental Table S1) were biochemically fractionated according to solubility using non-ionic and ionic detergents at different concentrations.…”

“…Homogenization of Brain Tissue-The brain extraction procedure was performed as described by Ö hrfelt et al, with minor modifications (22). Briefly, 100 Ϯ 10 mg of brain tissue was homogenized on ice in 1 ml of Tris-buffer (10 mM Tris-HCl, pH 6.8) containing complete protease inhibitor (Roche Diagnostics GmBH).…”

“…Immunoprecipitation-The immunoprecipitation of brain tissue extracts was performed according to Ref. 22, with minor modifications. Briefly, an aliquot (1 g) of the mouse monoclonal antibody SP12 (1 g/l), the mouse monoclonal antibody SMI81 (1 g/l), or IgG from murine serum (1 g/l, Sigma-Aldrich) (a negative control) was separately added to 100 l of magnetic Dynabead M-280 Sheep anti-mouse IgG (Invitrogen) and incubated for 1 h on a rocking platform at room temperature.…”