Proteome discoverer software

Proteome Discoverer (PD) software (2.1, Thermo Fisher Scientific) was used to perform database searching against the database containing the Uniprot Human Proteome (ID: UP000005640, downloaded on 25 May 2016), using both Sequest and Mascot search engines. Searches were performed with the following settings: a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.02 Da. Digestion by trypsin and two missed cleavage sites were allowed. Carbamidomethyl was defined as a fixed modification and phosphorylation (serine, threonine, tyrosine) and oxidation (methionine) were dynamic modifications. The results were filtered with the following parameters: only high-confidence peptides with a global false discovery rate (FDR) < 1% based on a target–decoy approach and first ranked peptides were included in the results.
The MS/MS results (RAW data) together with the PD results were inspected in a quality control (QC) analysis using an in-house-developed QC analysis (set of functions) in R as previously described [35 (link)]. Only the samples that passed the QC were used in the biomarker discovery study. Samples that generated low quality data were re-analyzed. If for several reasons samples did not meet the requested QC parameters, for example, reproducibility of retention time and mass calibration, these samples were excluded for further data analysis steps.

The mass spectra were filtered using Proteome Discoverer (PD) software (version 1.4.0.288, Thermo Fisher Scientific Inc., MA, United States). The spectrum extracted by PD was searched by Mascot (version 2.3.2, Matrix Science Ltd., London, UK). After the search, quantitative analysis was performed via the PD software according to the search results of Mascot and the spectrum after the first step of screening.

Protein preparation, LC-MS/MS analysis, and a database search for the identification of proteins were essentially performed as previously described (62 (link)), except for the following changes. The LC gradient elution was as follows: 0 min at 4% eluent B, 5 min at 5% eluent B, 30 min at 8% eluent B, 60 min at 12% eluent B, 100 min at 20% eluent B, 120 min at 25% eluent B, 140 min at 35% eluent B, 150 min at 45% eluent B, 160 min at 60% eluent B, 170 to 175 min at 96% eluent B, and 175.1 to 200 min at 4% eluent B. Mass spectrometry analysis was performed on a QExactive HF instrument (Thermo Fisher Scientific) at a resolution of 120,000 FWHM for MS1 scans and 15,000 FWHM for MS2 scans. Tandem mass spectra were searched against the UniProt database (7 August 2018; https://www.uniprot.org/proteomes/UP000002530) of Neosartorya fumigata (Af293) and the human protein sequences of azurocidin, cathepsin G, and RBP7, using Proteome Discoverer (PD) software (version 2.2; Thermo Fisher Scientific) and the algorithms of Sequest HT (a version of PD software [version 2.2]) and MS Amanda (version 2.0) software. Modifications were defined as dynamic Met oxidation and protein N-terminal acetylation as well as static Cys carbamidomethylation.

Raw files were, respectively, searched against rat and mouse protein databases (rat RefSeq database downloaded August 2015 containing 42,925 sequences, mouse RefSeq database downloaded October 2014 containing 58,513 sequences) using the SEQUEST algorithm embedded in Proteome Discoverer (PD) software (Thermo Scientific, version 1.4). Precursor mass tolerance was set as 10 ppm, and fragment mass tolerance was set as 0.02 Da. Number of maximum miss cleavage sites was set to 2. Carbamidomethylation of cysteine was set as static modification. N-terminal acetylation, methionine oxidation, as well as phosphorylation of serine, threonine, and tyrosine were included as variable modifications. False discovery rate (FDR) was calculated using Percolator. Only rank 1 and high confidence (with a target FDR q-value below 0.01) peptides were included in the final results. Proteome GO-term molecular function analysis was performed using the Automated Bioinformatics Extractor¹ (ABE) (35 (link)).