Computational protocol: Comparative proteomics of Rhizopus delemar ATCC 20344 unravels the role of amino acid catabolism in fumarate accumulation

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Protocol publication

[…] LC-MS/MS analysis was performed at the Radboud Proteomics Centre as described previously (). Measurements were performed by nanoflow reversed-phase C18 liquid chromatography (EASY nLC, Thermo Scientific) coupled online to a 7 Tesla linear ion trap Fourier-Transform ion cyclotron resonance mass spectrometer (LTQ FT Ultra, Thermo Scientific). The LC-MS/MS spectra obtained were identified and quantified using the maxQuant software (). The peptides were mapped against the in silico proteomes of R. delemar ATCC 20344 (obtained from the transcriptomics experiment) and RA 99-880 (obtained from Genbank, Project ID: 13066 ()) with the default settings, described in (). Only proteins with 2 or more unique peptide hits were considered for further analysis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE () partner repository with the dataset identifier PXD004600. [...] Metabolic enzymes were annotated using PRIAM (), and subsequently assigned to KEGG (; ) pathways (see for details on the KEGG pathway mapping). Enrichment analysis of differentially expressed pathways was performed using the hypergeometric test implementation (“phyper”) of the R software environment (). We used the identified proteins that could be mapped to a KEGG pathway as the universe (with size N = 277). Note that the terms “differentially expressed” and “overexpressed” refer to differences in relative protein abundances, and denote a fold-change of 1.5 and 2 as lenient and stringent thresholds. […]

Pipeline specifications

Software tools MaxQuant, PRIAM
Databases ProteomeXchange KEGG KEGG PATHWAY
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Rhizopus oryzae
Chemicals Amino Acids, Nitrogen, Urea