Proteinlynx global server plgs software

The mass spectrometry system used for the proteomic approach was a nanoAcquity UPLC-Xevo QTof MS system (Waters, Manchester, UK), using the ProteinLynx Global SERVER (PLGS) software (Waters, Milford, MA, USA), as previously described by [69 (link)] after downloading Uniprot database. The PLGS software applied the Monte-Carlo algorithm to obtain the difference of protein expression between the groups, considering p < 0.05 for downregulated proteins and 1 − p > 0.95 for upregulated proteins. After the identification and categorization of proteins, the Cytoscape 3.6.1 (Java^®) software (National Institute of General Medical Sciences, Rockville, MD, USA) was used for bioinformatics analyses with the ClueGO plugin for the determination of biologic processes groups [70 (link)].

Samples (10 µl) were injected into the C18 UPLC column (Waters UPLC system). The separation of all the samples was performed on ACQUITY UPLC BEH C18 column (Waters, UK) (75 µm × 150 mm × 1.7 µm). A gradient elution program was run for the chromatographic separation with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) followed by analysis on the Q-TOF instrument for MS and MS/MS [Synapt G2 Mass Spectrometer equipped with an electrospray ionization (ESI) source]. Sample analysis was performed in the positive mode. The experimental instrument parameters include polarity- ES+, analyser- resolution mode, capillary (kV)- 3.5000, source temperature- 150°C, sampling cone- 45, extraction cone- 4.5, source gas flow- 30 mL/min, desolvation temperature- 350°C, cone gas flow- 30 L/Hr and desolvation gas flow- 800 L/Hr. The TOF MS setup includes a Da range from 50 to 2,000 Da and a scan time of 0.5 s. The raw data were processed by Waters MassLynx 4.1 peptide editor software to get the complete integrated sequence of the sample. The individual peptide MS/MS spectra were matched to the database sequence with the help of the ProteinLynx Global Server (PLGS) software (Waters). The peptides were loaded with buffer A and eluted with buffer B (95% acetonitrile, 0.1% formic acid) at a flow rate of 0.3 ml/min.