Fig 1.
Venn diagram of proteins identified in NETs.
A. Venn diagram of overlapped and unique NETs proteins identified by means of mass spectrometry. Number of proteins in the overlapping and not-overlapping areas is shown. B. Venn diagram of NETs proteins grouped on the basis of database annotation. Number of proteins in the overlapping and not-overlapping areas is shown.
Fig 2.
Pie chart of cellular component gene ontology annotation of NETs samples.
Fig 3.
Spearman’s correlogram associated to unsupervised hierarchical clustering analysis of NETs.
In Spearman’s correlogram the coefficient values are depicted by a pseudo color scale from 0.8 (blue) to 0.9 (red); moreover the tree dendrogram displays the results of an unsupervised hierarchical clustering analysis placing similar Spearman's coefficient values near each other.
Fig 4.
Volcano plot of univariate statistical analysis of NETs samples.
Volcano plot based on fold change (Log2) and P value (-Log10) of all the proteins identified in PMA (4a), LPS (4b) and A23187 (4c) as compared with spontaneous NETs. White circles indicate the proteins displaying statistically significant changes.
Fig 5.
Heat map of NETs highlighted proteins.
Heat map of proteome profile of 25 proteins highlight through the combined use of univariate statistical analysis, support vector machine and partial least square discriminant analysis. In heat map, each row represents a protein, and each column corresponds to each condition. Normalized Z-score of proteins abundance are depicted by a pseudo color scale with red indicating positive expression, white equal expression, and blue negative expression compared to each proteins values, whereas the tree dendrogram displays the results of an unsupervised hierarchical clustering analysis placing similar proteome profile values near each other. Visual inspection of the dendrogram and heat map demonstrate the possibility to distinguish among the different condition of NETs induction.
Fig 6.
Heat map of NETs gene ontology enrichment analysis.
Heat map of biological process enrichment of different NETs stimuli compared to no stimulated condition. In the heat map, each row represents a protein, and each column corresponds to each condition. Normalized Z-score of proteins abundance are depicted by a pseudo color scale with red indicating positive expression, white equal expression, and blue negative expression compared to each proteins values, whereas the tree dendrogram displays the results of an unsupervised hierarchical clustering analysis placing similar proteome profile values near each other. Visual inspection of the dendrogram and heat map demonstrate the ability of these biological processes to distinguish between the A23187 and PMA/LPS stimuli.
Fig 7.
Venn diagram of total NETs peptides with at least one post translational modification identified by means of mass spectrometry.
Venn diagram shows overlapped and unique peptides. Numbers represent the distinct peptides/post translational modifications in the overlapping and not-overlapping areas.
Fig 8.
Volcano plot of univariate statistical analysis of spontaneous and induced NETs.
Volcano plot based on fold change (Log2) and P value (-Log10) of all the peptides with at least one PTM identified in PMA (8a), LPS (8b) and A23187 (8c) as compared with spontaneous NETs. White circles indicate the peptides displaying statistically significant changes.
Fig 9.
Heat map of highlight peptides.
Heat map of peptide profile of NETs samples. In heat map, each row represents a modified peptide, and each column corresponds to each stimulus. Normalized Z-score of peptides abundance are depicted by a pseudo color scale with red indicating positive expression, white equal expression, and blue negative expression compared to each peptide value, whereas the tree dendrogram displays the results of an unsupervised hierarchical clustering analysis placing similar peptide profile values near each other.
Fig 10.
Three-dimensional (3D) model of MPO.
3D-model of MPO based on its crystal structure (PDB codes: 5 MFA). The model is colored in function of second structure i.e. gray, red and cyan respectively for coil, alpha helix and beta-strand. In yellow methionine sulfoxide is shown.