title: Heat-mediated enrichment of α-synuclein from cells and tissue for assessing post-translational modifications.
abstract: α-Synuclein (α-syn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of α-syn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward α-syn PTMs. Here, by taking advantage of α-syn thermostability, we applied a method to achieve high enrichment of soluble α-syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% α-syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of α-syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies.
authors.affiliation: Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal.
authors.forename: Hugo
authors.initials: H
authors.lastname: Vicente Miranda
authors.forename: Wei
authors.initials: W
authors.lastname: Xiang
authors.forename: Rita M
authors.initials: RM
authors.lastname: de Oliveira
authors.forename: Tânia
authors.initials: T
authors.lastname: Simões
authors.forename: José
authors.initials: J
authors.lastname: Pimentel
authors.forename: Jochen
authors.initials: J
authors.lastname: Klucken
authors.forename: Deborah
authors.initials: D
authors.lastname: Penque
authors.forename: Tiago F
authors.initials: TF
authors.lastname: Outeiro
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009566:Nitrates / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009566:Nitrates; D051844:alpha-Synuclein; D014357:Trypsin
keywords: Parkinson's disease; post-translational modifications; proteomics; α-synuclein