Computational protocol: Identification of TTAGGG-binding proteins in Neurospora crassa, a fungus with vertebrate-like telomere repeats

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Protocol publication

[…] The raw data was processed with MaxQuant [] using the Neurospora version 3 database (9873 entries) with standard settings except the following (FastLFQ was deactivated and match between runs was activated). The results including LFQ (label-free quantitation) intensities were loaded into Perseus (version Protein groups consisting of only identified by site proteins, reverse hits and contaminants were removed. Proteins that were detected in three of the four TTAGGG replicate measurements were considered. Missing values were imputed in each column separately by using a normal distribution with a width of 0.3 downshifted by 1.8 (standard values). To obtain the LFQ enrichment, the difference of the arithmetic mean of the quadruplicates of control and bait was calculated and plotted (x-axis). A two-sided t-test with 250 randomizations was performed between the four replicates of the telomeric bait against the four replicates of the control baits. A significance threshold was determined at an FDR of 0.05 and s0 = 0.2. The results were exported and plotted in R (version [...] Annotated N. crassa peptide sequences of the 12 hits were blasted using the NCBI blastp (protein-protein BLAST) standard search. Searches were carried out against S. cerevisiae (taxid:4932), S. pombe (taxid:4896), A. fumigatus (taxid:746128) and C. albicans (taxid:5476). Blast hits were reverse blasted against N. crassa (taxid:5141) as search organism to verify homology. Homologs were only considered valid if both the forward and reverse blast yielded e-values smaller than e-8 and if in both directions the respective proteins were each the strongest hit. In some cases also weak homologs (e-values smaller than e-3) are reported, in which the forward and/or reverse blast identified a different protein as the direct homolog. […]

Pipeline specifications

Software tools MaxQuant, Perseus, BLASTP, DELTA-BLAST
Applications MS-based untargeted proteomics, Amino acid sequence alignment
Organisms Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe
Chemicals Zinc