Article Figures & Data
Figures
- Fig. 1.
Post-translational modifications of α-syn. A, the locations of the main α-syn PTMs (phosphorylation, ubiquitination, nitration, and truncation) are shown. Disease-associated PTMs identified in Lewy bodies are shown in the upper part of the scheme, and those identified from in vitro studies are shown below. Green bars above and below the sequence of α-syn show epitope maps of the most frequently used non-PTM and non-oligomer-specific antibodies for immunoassay-based α-syn quantification in the CSF and blood plasma. Stars indicate putative or incomplete epitope mapping. B, dot blot showing evidence that PTMs can interfere with the detection of modified α-syn by antibodies. Using the Syn-211 clone, the sensitivity for pY125 α-syn was reduced, and double-phosphorylated α-syn (pY125/pS129) was nearly undetected. The antibodies used for detection are shown on the right-hand side of the blots. C, schemes depicting the ordinary, α-syn monomer-directed ELISA assays (left) and the principle of the oligomer-specific ELISA system (right). Adapted from Ref. 23.
- Fig. 2.
Epitope mapping of α-syn antibodies. Monoclonal and polyclonal antibodies are shown in green and red, respectively. Residue numbers corresponding to the epitopes are shown in bold (when available).
- Fig. 3.
Chemical protein synthesis as a tool to generate well-defined full-length protein standards for absolute quantification. A, mechanism of the native chemical ligation reaction, a core component of chemical protein synthesis approaches. B, expressed protein ligation can be combined with enzymatic modifications using specific enzymes, such as PLK3, thus allowing the preparation of α-syn modified at multiple residues to enable investigation of how single or multiple PTMs influence α-syn detection by different antibodies and comparison of the performance of mass-spectrometry-based quantification to the immunoassays currently in use. Adapted from Ref. 58. C, total chemical synthesis of α-syn using three fragments, all produced via chemical peptide synthesis, allowing the introduction of PTMs and/or various labels (shown as colored stars) anywhere within the α-syn′ sequence (whereas semisynthesis, shown in B, is mostly restricted to PTMs within the C or N terminus of α-syn). The central fragment (green) initially bears a temporary thiazolidine (Thz) protection to prevent the peptide from polymerizing and/or undergoing cyclization. Adapted from Ref. 131. D, immunocytochemistry (left) and Western blot (right) show that pY125 is a very labile modification, highlighting that the semisynthetic pY125 α-syn standard can be very useful for developing protocols to stabilize this modification in biological samples.
- Fig. 4.
Overview of the main workflow from sample preparation to MS analysis. The diagram outlines the required workflow for sample preparation prior to MS analysis and quantification via selected reaction monitoring (SRM). Total protein extraction is typically carried out by means of SDS followed by protein equalization using a BCA assay. Prior to processing, protein extracts are spiked with a full-length heavy isoform 15N α-syn protein carrying the PTM of interest such as nitration (3NT) or phosphorylation at tyrosine or serine residues. Samples are typically fractionated via isoelectric focusing (IEF), SDS-PAGE, or size-exclusion chromatography (SEC). Following fractionation, samples are digested by trypsin (Tryp), Asp-N, cyanogen bromide (CNBr), or Glu-C using consecutive or combined digestions. Gel bands are extracted at the migration height of α-syn protein and subjected to individual proteolysis. For combined proteolytic analysis, such as in the case of Tryp and Glu-C digestions, samples are run in duplicate and digested separately or sequentially as in the case of Tryp and Asp-N proteolysis. Phosphopeptide enrichment (i.e. using titanium dioxide (TiO2)) chromatography allows for the enrichment of pathologically relevant phosphorylated peptides such as at residues p-S87, p-Y125, and p-S129. If full-length protein standards are not available, then the use of synthetic heavy-labeled surrogate peptides (AQUA) allows for individual spiking of reference peptides bearing PTMs of particular interest. Following proteolysis and reference peptide spiking, samples are analyzed via liquid chromatography (LC) coupled with SRM.
- Fig. 5.
Proteolytic peptides typically observed via mass spectrometry following trypsin (A), Glu-C (B), cyanogen bromide (C), and Asp-N (D) digestion. (MS preferential peptides are highlighted in red.) E, liquid chromatography MS spectrum of a typical LC-SRM analysis of α-syn tryptic and Glu-C peptides derived from rat nigral tissue. Endogenous rat syn (black) and viral induced overexpressed human α-syn (red) can be quantified during a single LC-SRM run. F, zoom of the highlighted area in E showing the total ion extraction chromatogram of tryptic fragment 81–96 with its heavy-labeled α-syn isoform (15N) below. A shift in retention time can be observed between rat and human isoforms accounting for the difference in sequence at residue 87 or for the phosphorylation occurring at p-S87, whereas the retention time of surrogate peptides (15N heavy isoform) corresponds to the endogenous form.
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