Phosphoproteomics of Peptide Mixtures

Peptide mixture was analysed by an Ultimate® 3000 Nano-LC systems (Thermo Scientific, USA) coupled with a microTOF-Q II (Bruker, Germany). The acquisition was controlled by HyStar™ version 3.2 (Bruker, Germany). MS and MS/MS spectra covered the mass range of m/z 400–2000 and m/z 50–1500, respectively. The raw data format (.d) files were processed and converted to mascot generics files (.mgf) using Compass DataAnalysis™ software version 3.4 (Bruker, Germany) and submitted for database searches using Mascot Daemon software (Matrix Science, USA) against in-house transcriptomics database. Miss cleavage was allowed at one. Variable modifications were set as carbamidomethyl (C), oxidation (M), phospho (ST), and phospho (Y), MS peptide tolerance was 0.8 Da and MS/MS tolerance was 0.8 Da. Differential phosphoproteins were classified by gene ontology using Blast2Go software. Protein domains were predicted by Pfam 32.0 (September 2018, 17929 entries). Swiss model server was used for three-dimensional (3D) structure modeling^{71 (link)}. The template was selected by a sequence with the highest percentage identity. The.pdb file of modeled protein structures were downloaded and analyzed by Visual Molecular Dynamics software⁷² .