Peakview 2

Peakview 2.2 software (SCIEX, Framingham, MA, USA) was used for data processing and to record the retention time and masses of the detected molecules (Tsugawa, Cajka et al. 2015). The detected masses ranged from 50 to 1000 Da. Metabolites were assigned tentatively by matching mass spectral data of the identified metabolites in both ionization modes with reported data provided in online libraries and databases (Human Metabolome Database and Pubchem), alongside retention times and fragmentation patterns comparison to reference standards whenever possible.

The mass spectrometry data obtained were processed using PeakView® 2.2 Software (SCIEX, Foster City, CA, USA) and exported as mgf files. Proteomics data analysis were performed by using four search engines (Mascot Server v.2.6.1, OMSSA, X!Tandem, and MyriMatch) and a target/decoy database built from sequences in the Homo sapiens reference proteome at UniProt Knowledgebase. Search parameters included: trypsin as enzyme; two allowed missed cleavages; carbamidomethyl (C) as a fixed modification and acetyl (Protein N-term) and Oxidation (M) as variable modifications; peptide mass tolerance ± 25 ppm for precursors and 0.02 Da for fragment masses. Score distribution models were used to compute peptide-spectrum match p-values, and spectra recovered by a false discovery rate (FDR) <= 0.01 (peptide-level) filter were selected for quantitative analysis (Li et al., 2017a (link)).

All data analysis steps were performed separately for the whole proteome and secretome samples. A database search was conducted in ProteinPilot 5.0.2 software (SCIEX, Framingham, MA, USA) against the UniprotKB D. solani D s0432-1 database (version from 19.10.2020) with an automated false discovery rate analysis. Only proteins identified at 1% FDR were considered valid identifications. SWATH-MS data were analyzed in a similar manner as described before [114 (link)]. Database search results were imported to the PeakView 2.2 software (SCIEX, Framingham, MA, USA) to construct spectral libraries with the exclusion of shared peptides. SWATH-MS measurement files were uploaded into software and analyzed using relevant libraries. The results were exported to MarkerView software (SCIEX, Framingham, MA, USA) and normalized using the Total Area Sums (TAS) approach. T-tests between studied groups were performed, and the protein concentration changes were considered to be statistically significant if the p-value was lower than 0.05 and the fold change was lower than 0.5 or greater than 2.
The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE [115 (link)] partner repository with the dataset identifier PXD028047.

Raw data were acquired by Analyst 1.7.1 software (SCIEX, Foster City, CA, United States) and checked/viewed with PeakView 2.2 software (SCIEX, Foster City, CA, United States). Metabolites search and prediction were performed by MetabolitePilot™ 2.0.4 software using default settings (SCIEX, Foster City, CA, United States).

To check the absence of carry-over effects and to control the stability of recorded fingerprints, blank and quality control (QC) matrix samples were analyzed within UHPLC-HRMS/MS sequences. The in-batch sequence of tested samples was random to avoid any possible time-dependent changes during analysis, which could result in false clustering. To control the overall performance of the instrumental system, QC samples were inserted into the sequence, always after a set of ten tested samples, and analyzed under the same conditions. The QC sample was prepared as a mixture of all analyzed samples. The good instrument performance was documented by a tight clustering of these QC samples (i.e., similarity of their fingerprints) in the principal component analysis (PCA) score plot. The instrument control was carried out with the Analyst TF 1.7.1 software (SCIEX, Canada) and PeakView 2.2 software (SCIEX, Canada).