Computational protocol: Quantitative proteomics in A30P*A53T α synuclein transgenic mice reveals upregulation of Sel1l

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Protocol publication

[…] The sample was then fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column. Briefly, peptides were first separated with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate pH 10 over 80 min into 80 fractions. Then, the peptides were combined into 18 fractions and dried under vacuum. Peptides were dissolved in 0.1% FA (Formic acid, Fluka) and directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient was composed of an increase from 6% to 85% solvent B (0.1% FA in 98% ACN), all at a constant flow rate of 300 nl/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed by a Q Exactive™ Plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific).The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive™ Plus (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using the NCE setting at 27, 30, 33; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 2.0E4 in the MS survey scan with a 30.0-s dynamic exclusion.The resulting MS/MS data were processed using Mascot search engine (v.2.3.0). Tandem mass spectra were searched against the SwissProt_Mouse database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. The mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethyl on Cys was specified as a fixed modification and oxidation on Met was specified as a variable modification. For the protein quantification method, TMT-6plex was selected in Mascot. FDR was adjusted to < 1%, and the peptide ion score was set > 20. [...] Some identified proteins that appear in some samples but not in others were removed. Gene Ontology (GO) annotation of the proteome was performed using the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Cluster membership was visualized by a heat map using the "heatmap.2" function from the "gplots" R-package. There, we used wolfpsort a subcellular localization prediction software, to predict subcellular localization. If some identified proteins were not annotated by the UniProt-GOA database, then the InterProScan software was used to annotate the protein's GO function based on the protein sequence alignment method. Proteins were classified by Gene Ontology annotation based on three categories: biological process, cellular component and molecular function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate the protein KEGG pathway by a two-tailed Fisher's exact test. […]

Pipeline specifications

Software tools Mascot Server, gplots, InterProScan
Databases UniProt-GOA KEGG KEGG PATHWAY
Applications MS-based untargeted proteomics, Amino acid sequence alignment
Organisms Mus musculus, Homo sapiens
Diseases Neurodegenerative Diseases, Mitochondrial Diseases