mascot software version 2 6  (Matrix Science)

Journal: The American Journal of Pathology

Article Title: The Parkinson-Associated Toxin Paraquat Shifts Physiological α-Synuclein Tetramers toward Monomers That Can Be Calpain-Truncated and Form Oligomers

doi: 10.1016/j.ajpath.2023.01.010

Figure Lengend Snippet: Calpain 1–truncated and oligomeric α-synuclein (αS) in tetramer-abrogating 3K mouse brain. A and E: Recombinant soluble (rec sol) or fibrillized αS (rec fibr) cleaved with calpain 1 (for 75 minutes) and wild-type (WT) or 3K αS mouse brain extracts were subjected to SDS gel electrophoresis and Coomassie staining and blotting to identify gel-slicing sites targeting αS oligomers (approximately 70 kDa) and α-synuclein truncation (αS-Δ; 10 to 12 kDa) in mouse brain (indicated by ] ; A ) and analyzed by mass spectrometry ( E ). B: Cortical extracts show full-length, truncated, and αS oligomers in 1K and 3K versus non-transgenic (Ntg) and WT mouse brain. The difference in migration of the human αS is due to the additional positive charges in the K-mutant protein. C: Quantification of truncated versus full-length αS signal in similar expressing WT, 1K, and 3K cortices using the 105 αS antibody that detects full-length and calpain-truncated αS. 3 KL (lower-expressing 3K αS mouse line) and 3K display similar increase in αS-Δ versus 1K and WT. D: Hypothetical model based on data herein. A shift of native αS tetramers to more protease-accessible monomers by familial Parkinson disease (PD) mutations and the amplification of the E46K mutant (E35 + E46 + E61). Paraquat, an environmental toxin linked to a higher PD risk, can also trigger the tetramer/monomer shift, and aberrant activation of calpain 1 results in monomer truncation, which, in turn, promotes aggregates distinct from physiological tetramers. E: Tryptic peptides from liquid chromatography coupled with tandem mass spectroscopy spectra were analyzed using Mascot software version 2.6.2 (Matrix Science Inc., Boston, MA). Green letters are sequences of the human α-synuclein protein that are detected with high confidence. Each underline represents an independent data point for peptide identification. Red underline is modification at a given amino acid. n = 3 per genotype ( C ). ∗ P < 0.05 (one-way analysis of variance post hoc Tukey test). N-acet., N-acetylated.

Article Snippet: E: Tryptic peptides from liquid chromatography coupled with tandem mass spectroscopy spectra were analyzed using Mascot software version 2.6.2 (Matrix Science Inc., Boston, MA).

Techniques: Recombinant, SDS-Gel, Electrophoresis, Staining, Mass Spectrometry, Transgenic Assay, Migration, Mutagenesis, Expressing, Amplification, Activation Assay, Liquid Chromatography, Tandem Mass Spectroscopy, Software, Modification