Figure 4

Label-free quantitative proteomic analysis in an α-Gal A-depleted human podocyte cell culture model shows an increase of lysosomal proteins in both cell culture models. (A) Proteome analysis of control podocytes and α-Gal A-depleted podocytes (shRNA894). MS2 spectrum of the identified GLA peptide (P06280, residues 333–356 QGDNFEVWERPLSGLAWAVAMINR) which was identified only in control samples but not in knockdown samples. (B) Volcano plot showing logarithmized fold changes of label free quantification (LFQ) values in knockdown vs. control podocyte samples. Log2 ratios of shRNA over control are plotted against the negative logarithmic P value of the Student’s t-test. Each dot represents a protein (FDR = 0.2, s0 = 0.1). Significant proteins are marked in blue with their respective gene symbol (n = 6). (C) Scatter plot analysis of fold changes in podocytes and urine-derived cells with proteins of interest displayed in blue. Log2 fold changes of the podocyte GLA knockdown model (shRNA/control) are plotted against the Log2 fold changes of the original primary urine cells (Fabry vs control). Proteins regulated positively in both datasets are marked with their respective gene symbol. (D) Scatterplot of 2D GO enrichment analysis in podocytes and urine-derived cells. Fold changes of both datasets were subjected to dimensionality reduction using 2D GO enrichment algorithm (FDR 0.05). Differently distributed GO terms are in blue. The GO-CC term “lysosomal lumen” is marked in black. Uniprot keywords related to the disease mechanism (Glycosidase, Glycoprotein) are labeled in red. (E) Mapping of lysosomal lumen proteins on initial scatter plots. Lysosomal lumen proteins are marked in black.