Wishart, Thomas. (2021). Proteomic correlates of vulnerability in the presymptomatic synapse of a mouse model of SMA and a rat model of ALS Type 8, [dataset]. University of Edinburgh. Roslin Institute. https://doi.org/10.7488/ds/3124.
Data Generation: Tandem-mass-tagged mass-spectrometry (TMT-MS/MS) analyses of hippocampal synaptosomes of the Taiwanese mouse model of SMA, generated from n=5 each wildtype (Smn+/+), heterozygous (Smn+/-; SMN2tg/0) and homozygous (Smn-/-; SMN2tg/0) animals, respectively, yielded 6660 raw protein identifications derived from peptide sequences searched against Mus musculus UniProtKB/Swiss-Prot sequences using the MASCOT Search Engine (V2.3.2) through Proteome DiscovererTM software (Version 1.4, Thermo Fisher). Filtering to eliminate protein sequences identified by fewer than 2 or more unique peptides produced 6195 identifications ready for subsequent analysis.
TMT-MS/MS analyses of hippocampal synaptosomes against a novel rat model of ALS 8, generated from n=5 each wildtype (VAPB+/+), heterozygous (VAPB P56S/+) and homozygous (VAPB P56S/P56S), respectively, yielded 7828 raw protein identifications derived from peptide sequences searched against Rattus norvegicus UniProtKB/Swiss-Prot sequences using the MASCOT Search Engine (V2.3.2) through Proteome DiscovererTM software (Version 1.4, Thermo Fisher). Filtering to eliminate protein sequences identified by fewer than 2 or more unique peptides produced 5942 identifications ready for subsequent analysis.
Generation of ratios comparing heterozygous and homozygous expression compared to wildtype of filtered proteins enabled normalised measurement of expression alterations for subsequent analyses. Expression of each protein at wildtype is therefore equivalent to 1 (ie. wildtype: wildtype), while values >1 at heterozygote or homozygote indicate a relative increase in expression, and values <1 indicate a relative decrease in expression.
Application of Markov Clustering algorithm set to a Pearson correlation coefficient of 0.98 to each >2 peptide dataset, inputted as an identifier column of UniProt/Swiss-Prot Accession Number and expression ratio values at wildtype, heterozygous and homozygous in each condition produced discrete clusters based on the sole parameter of similarity in expression profile alteration through genotype. Isolated clusters correlating with levels of full-length Smn expression (SMA model) or mutant VapbP56S expression (ALS 8 model), constituting n=3802 proteins in SMA model and n=3168 in ALS 8 model were mapped to human gene identities in the Ingenuity Knowledge Database (Qiagen).
Ethics: Animals and tissue harvesting
All animals used in this project were treated in accordance with the guidelines outlined by the UK Animal Scientific Procedures Act (1986), adhering to the three principles of Reduction, Refinement and Replacement.
Description of data files:
-Supplementary Table 1_Analysis-Ready_Mapped Clusters_SMA
-Supplementary Table 2_Analysis-Ready_Mapped Clusters_VAPB
Datasets (each in two formats_.xls & .xlsx) contain all biologically relevant alterations identified through expression cluster analysis. 1 for each condition. Alteration of protein expression by >20% in homozygote synapse compared to through colourisation in green (decrease relative to wildtype) or red (increase relative to wildtype). Intensity of colour corresponds to magnitude of change.
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