Mascot daemon 2

MS/MS spectra were assigned to peptide sequences by the MASCOT Daemon 2.3.2 search engine (Matrix Science) in follow-up mode using first the contaminant database described by Pereira et al. (51) including the 23 most abundant proteins from fetal calf serum, and then the SwissProt Homo sapiens database (20,396 polypeptide sequences). Standard search parameters included: trypsin as a proteolytic enzyme with two possible miss-cleavages at maximum, tolerances of 5 ppm and 0.02 Da for the MS and MS/MS signals, respectively, oxidation of methionine, deamidation of glutamine and asparagine, and acetylation of N-termini, as variable modifications, carbamidomethylation of cysteine as fixed modification, and a peptide p-value below 0.01. A protein was considered validated when at least two different peptides were detected, resulting in a protein identification false discovery rate below 1% as verified with a reverse decoy database search. To account for possible genome heterogeneity between patients, only peptides common between all patients (at least identified once in a patient sample) were used for the comparative proteomics. Spectral counts corresponding to the number of MS/MS spectra assigned per protein were used as a proxy for the abundance of the proteins in each condition, considering only unambiguous peptides (listed only once in the database).

Mass spectrometry data files were searched against a combined database of predicted D. citri, endosymbiont (Carsonella, Profftella, and Wolbachia), and CLas proteins using Mascot Daemon 2.3.2 (Matrix Science, Boston, MA). MS/MS search parameters included fixed modifications (cysteine: Methylthio), variable modifications (asparagine, glutamine: deamidated; methionine: oxidation), and maximum one missed cleavage. Thermo *.raw files were converted into Mascot Generic Format using MSConvert in Protewizard. Files with the *.dat extension were exported from Mascot and loaded into Scaffold Q+ 4.4.1.1 (Proteome Software, Portland, OR) and used to calculate normalized spectral count for each protein from each sample. Scaffold protein and peptide thresholds were set at 95%, with a minimum peptide number of two per protein–this resulted in a protein false discovery rate (FDR) of 2.9% and a peptide FDR of less than 0.09% [27 (link)],[28 (link)]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [29 (link)] via the PRIDE partner repository [30 (link)] with the dataset identifiers PXD003096 and PXD003097.

Spectral data were searched against a combined database described previously [14 (link)] containing predicted proteins from D. citri, its endosymbionts Carsonella, Profftella, Wolbachia as well as CLas using Mascot Daemon 2.3.2 (Matrix Science, Boston, MA). The database had a total of 33,112 combined insect and microbial sequences as well as 112 common contaminant sequences. The search parameters allowed for fixed methylthio modification and variable modifications (methionine oxidation; asparagine, glutamine deamidation) with a peptide mass tolerance of ±20ppm and fragment mass tolerance of ±0.6Da. A maximum of one missed cleavage was allowed. Raw files were converted to Mascot Generic Format files using MSConvert in Proteowizard and used as input files for Mascot Daemon. Peptides were identified at a 95% threshold with a 0.03% decoy false discovery rate (FDR). Proteins were identified with a 99% threshold and 0.6% decoy FDR with a minimum of 2 matching peptides. A Fisher’s Exact Test was employed to identify proteins significantly differentially expressed in CLas+ compared to CLas- samples using spectral counting. A P-value cutoff of 0.05 and 2-FC were applied.