Mascot daemon

Peak lists in .raw format were imported into Progenesis QI and LC-MS runs aligned to the common sample pool. Precursor ion intensities were normalized against total intensity for each acquisition. A combined peak list was exported in .mgf format for database searching against L. mexicana sequences appended with common proteomic contaminants (8365 sequences). MascotDaemon (version 2.5.1, Matrix Science) was used to submit the search to a locally-run copy of the Mascot program (Matrix Science Ltd., version 2.5.1). Search criteria specified: Enzyme, trypsin; Fixed modifications, Carbamidomethyl (C); Variable modifications, Oxidation (M); Peptide tolerance, 5 ppm; MS/MS tolerance, 0.5 Da; Instrument, ESI-TRAP. Search results were filtered to require a minimum expect score of 0.05. The Mascot .XML result file was imported into Progenesis QI and peptide identifications associated with precursor peak areas. Relative protein quantification was derived from unique peptide precursor ion intensities. Accepted quantifications were required to contain a minimum of two unique peptides. Statistical testing was performed in Progenesis QI and ANOVA-derived p values were converted to multiple test-corrected q-values using the Hochberg and Benjamini approach. Final quantification results were stripped of non-Leishmania spp. identifications for brevity.

Peak lists in the form of Mascot generic files (mgf files) were prepared from raw data using MS Convert (Proteowizard project) and sent to peptide match search on Mascot server v2.4.1 using Mascot Daemon (Matrix Science Ltd.).
Peak lists were searched against protein databases including typical proteomics contaminants such as keratins, etc. Tryptic peptides with up to two possible miscleavages and charge states +2, +3, +4 were allowed in the search. The following peptide modifications were included in the search: oxidized Methionine (variable), phosphorylated Serine/Threonine/Tyrosine (variable) and carbamidomethylated Cysteine (static). Data were searched with a monoisotopic precursor and fragment ion mass tolerance 10ppm and 0.6 Da respectively. Decoy database was used to validate peptide sequence matches. Mascot results were combined in Scaffold v4.4.0 (Proteome Software Inc) and exported to Excel (Microsoft Office) for further processing and comparisons.
In Scaffold, the peptide and protein identifications were accepted if probability of sequence match and protein inference exceeded 95.0% and 99% respectively. Protein probabilities were calculated in Scaffold by the Protein Prophet algorithm; proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony (Searle, 2010 (link)).

Compass DataAnalysis 4.0 software (Bruker Daltonics) was used for data processing. Generated MGF files were then merged per lane and used in a database search (SwissProt, version 20150612), using the Mascot Daemon application included in an in-house MASCOT server (version 2.5, Matrix Science, London, UK) with the following constraints: taxonomy = Homo sapiens (20207 sequences); enzyme = trypsin; missed cleavage = 1; peptide and fragment mass tolerance =  ± 0.3 Da; fixed modifications = carbamidomethyl (Cys); variable modifications = oxidation (Met).
Label-free quantitative analyses were performed on three biological triplicates using the spectral counting method based on normalised exponentially modified protein abundance index (emPAI), as described by Shinoda et al.^{59 (link)}.
To obtain a comprehensive description of the over-represented biological processes and functionally-related groups of proteins within our dataset, a Bioinformatic Gene Ontology analysis was performed using the on-line FunRich (Functional Enrichment analysis tool) software 3.0 (www.funrich.org). The default Homo sapiens genome was used as background.

The procedure used for this analysis has been described previously^{55 (link)}. hSP-A (25 μM) was incubated with acrolein (500 μM) at 37 °C for 4 h. After removal of the excess unreacted acrolein with a PD SpinTrap G25, the samples were reduced with 100 mM NaBH₄ for 3 h at room temperature, neutralised with 2 N HCl, and precipitated with chloroform/methanol. The precipitated protein was dissolved in 8 M urea and 50 mM NH₄HCO₃, reduced with 10 mM Tris(2-carboxyethyl)phosphine hydrochloride, and alkylated with 2-iodoacetamide. The alkylated sample was digested by using sequencing-grade modified trypsin (Promega) in the presence of 0.01% ProteaseMAX surfactant (Promega) for 1 h at 50 °C. The recovered peptides were then resolved by reverse-phase nano-LC (DiNa Nano LC system; KYA Tech, Tokyo, Japan) and then directly fractionated onto a MALDI target plate with α-cyano-4-hydroxycinnamic acid by a spotter (DiNa Nano LC system). MALDI-MS and MS/MS were performed on an ABSCIEX TOF/TOF 5800 system (AB Sciex, Tokyo Japan). The MS/MS data were processed and subjected to database searches by using MASCOT Daemon (Matrixscience).

Tandem mass spectra were extracted using the extract_msn script (Thermo Fisher Scientific, Altrincham, UK) executed in Mascot Daemon (version 2.2.2.; Matrix Science, London, UK). Peak lists were searched against the UniProt Arabidopsis thaliana database (version ARATH, 2013‐05). Trypsin was included as the cleavage enzyme and a maximum of one missed cleavage was allowed. A peptide mass tolerance of 5 ppm and a fragment tolerance of 0.5 Da was included. Carbamidomethyl of cysteine was included as a fixed modification, with oxidation of methionine and phosphorylation of serine, threonine and tyrosine residues included as variable modifications. Data were validated using Scaffold (version 4.0.5, Proteome Software, Portland, OR, USA). Phosphopeptide identifications were accepted if established with at least 95% probability at the peptide level and at least 50% probability at the protein level and a threshold peptide ID score of 20 was applied.

The precipitated proteins from the culture supernatants of wild-type S. suis 2 05ZYH33 and T4SS-deficient mutant strain (ΔvirD4-89K) were analyzed through LC-MS/MS to identify the putative proteins secreted via T4SS. Protein samples were separated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), pretreated with trypsin, and analyzed through LC-MS/MS by using UltiMate3000 RSLCnano liquid chromatography/Bruker maxis 4G Q-TOF. The resulting peptide mass fingerprints were compared against the ORFs of the genome of 05ZYH33 by using Mascot and Mascot Daemon software (Matrix Science); matches with P < 0.05 were considered with high confidence. Signal peptides were predicted using SignalP 4.1 server.