Mascot distiller software

Data were processed automatically using Mascot Distiller software (version 2.7.1.0, Matrix Science). Peptides and proteins were identified using Mascot (version 2.6) through concomitant searches against Swiss-Prot (Homo sapiens taxonomy, downloaded in November 2019), a classical contaminants database (homemade), and their corresponding reversed databases. Trypsin/P was chosen as enzyme, and a maximum of two missed cleavages was allowed. Precursor and fragment mass error tolerances were set to 10 ppm and 25 mmu, respectively. Peptide modifications allowed during the search were: (1) cysteine carbamidomethylation (fixed); (2) acetylation of the protein’s N-terminus (variable), and (3) methionine oxidation (variable). Proline software (version 2.1) [29 (link)] was used to merge data for all patients. After merging, results were filtered: conserving rank 1 peptide-spectrum matches with a minimal length of 7 amino acids and a minimal Mascot peptide score of 25. With these parameters, the False Discovery Rate (FDR) for peptide/spectrum match identifications was below 1% according to the target-decoy approach. A minimum of two peptides was required for a protein group to be identified. Proline was then used to perform MS1-based label-free quantification of the protein groups identified, based on their specific peptide abundances (Supp. Table 1).

For data processing and protein identification the Automated Proteomics Pipeline (APP) was used to analyse the MS data (Malm et al., 2014 (link)). APP automates the processing of proteomic tasks such as peptide identification, validation and quantitation from LC-MS/MS data and allows easy integration of multiple separate proteomic tools. The raw MS data file was first processed using the Mascot Distiller software (version 2.4.3.2, Matrix Science, London, UK). The resulting mgf files were subsequently converted into the mzXML file format using msconvert (Kessner et al., 2008 (link)). The data were searched against the Bgh genome using several search engines in parallel and the following settings: trypsin specific digestion with two missed cleavages allowed; peptide tolerance of 200 ppm; fragment tolerance of 0.5 Da; methylthio on Cys and iTRAQ 4-plex for peptide N-t and Lys used as fixed modifications; oxidized Met and Tyr for iTRAQ 4-plex analysis in variable mode. PeptideProphet was used to validate the results from the searches (Keller et al., 2002 (link)).

The acquired MS/MS data were preprocessed with Mascot Distiller software (v. 2.5/2.6, MatrixScience, London, UK) and a search was performed with the Mascot Search Engine (MatrixScience, London, UK, Mascot Server 2.5/2.6) against the Paramecium protein (40 460 sequences; 17901632 residues) database. To reduce mass errors, the peptide and fragment mass tolerance settings were established separately for individual LC–MS/MS runs after a measured mass recalibration, as described previously (41 (link)). The Mascot search parameters were as follows: mass tolerance for peptides: typically 5 ppm, mass tolerance for fragments: typically 0.01 Da, enzyme, Trypsin; missed cleavages, 1; fixed modifications, Methylthio (C); variable modifications, Oxidation (M); instrument, HCD. The FDR was calculated and results were filtered to acquire 1% false positive identifications. Details of all mass spectrometry experiments are listed in Supplementary Table S2.

Mass spectra were acquired in a positive-ion reflector mode with the use of a 4800 Plus MALDI-TOF/TOF Analyzer (Applied Biosystems, Framingham, USA). Alpha-cyano-4-hydroxycinnamic acid (CHCA) from Sigma-Aldrich (Munich, Germany), dissolved in 50:50 water/acetonitrile (J.T. Baker, Deventer, The Netherlands) with 0.1% TFA – final concentration (Sigma-Aldrich, Munich, Germany), was exploited as a MALDI matrix. External calibration was achieved with a 4700 proteomics analyzer calibration mixture provided by Applied Biosystems. Samples were spotted onto a 384 Opti-TOF MALDI plate and analyzed. Data Explorer Software, Version 4.9 was applied to process acquired spectra. Mascot Distiller Software (version 2.5.1.0, Matrix Science) was employed to predict fragment ions from given peptide sequences and overlay them on the acquired MS/MS spectra.

For protein expression analyses yeast were harvested during the exponential phase of growth at an OD₆₀₀ of 0.6, separated by filtration (see below) and cryogenically grinded in a SPEX Freezer Mill. The samples were resuspended in Y‐PER yeast protein extraction reagent (Thermo Fisher) and cellular debris removed by centrifugation. Each sample was chemically labeled separately with one of up to eight distinct isobaric iTRAQ reagents (Ross et al., 2004). Peak lists were extracted using the MASCOT Distiller software (MatrixScience). False discovery rates (FDR) were estimated by searching equivalent reversed or randomized sequence databases, using a cutoff value of 1% FDR. Protein‐level expression ratios were calculated using intensity‐weighted averages from matching peptides after outlier removal. This experiment was performed in duplicate. Comparing the two sample replicates, we fitted a Cauchy distribution using the EasyFit statistical package (MathWave, Inc.). Peptide p‐values were calculated using a two‐tailed t‐test.

Briefly, cellular protein (1 μg/μl) was mixed with 100 μl acetone and centrifuged at 15,700 g for 10 min. After centrifugation, the supernatant was discarded and the protein residues were kept and evaporated to dry. Protein residues were re-dissolved with 18 μl 25 mM ammonium bicarbonate aqueous solution, reduced and alkylated, and then sequence-grade trypsin (Promega) 2 μl was added and digested at 37°C for 16 hrs. After digestion, tryptic peptide solution was injected into the nano LC system, precursor ions of peptides were fragmented by collision gas to obtain tandem MS (MS/MS) spectra and detected by the high resolution linear ion-trap (LTQ) Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Raw data files (which contained precursor and fragment ions) were processed with Mascot Distiller software (Matrix Science Inc., Boston, MA, USA) to create the peak lists which were uploaded to the Mascot server (Matrix Science Inc.) for protein identification.