Download Supplementary File S2: analysis of protein-protein

Supplementary File S2: In silico analysis of highly upregulated proteins on osteosarcoma.
To investigate protein-protein-interactions (PPIs), protein clustering, networks, subcellular localisation of the proteins in our hit list and biological processes
wherein the proteins are involved, we used slightly less stringent criteria for protein selection than the criteria that were applied to obtain the proteins included
in Table 1 of the main manuscript. We filtered proteins based on their (1) expression in 4/5 OS cell lines, (2) >3-fold upregulation on OS cells vs. hp-OBs and
(3) with p<0.05 significance. This retrieved 123 highly upregulated proteins from our dataset which were entered into STRING 9.0 (www.string-db.org) using
default settings. Figure 1 shows a PPI map of physical interactions between upregulated surface proteins on OS.
Figure 1: Protein-protein Interaction map of 123 significantly upregulated surface proteins on OS compared to healthy bone cells. This PPI map was
generated by STRING 9.0. Thicker lines represent stronger associations. The squares highlight functional clusters identified within the STRING network,
based on molecule density. The inlays depict these functional clusters schematically (analysed with ClusterONE in Cytoscape).
Figure 1
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In this map, we can identify 6 high density clusters, which are enlarged and depicted schematically in the inlays in Figure 1. These clusters contain (1) an
integrin network connected to several growth factor receptors (MET, FGFR1 and -2); (2) a cluster containing several Ephrins and Ephrin Receptors (EFNB2
and -3, EFNA5, EPHA2 and EPHB2 and -4); (3) a cluster of transporter molecules; (4) a cluster containing both the insulin (INSR) and a insulin growth factor
receptor (IGF-1R) and a tumour necrosis factor receptor (TNFRSF10A); (5) a slightly comparable cluster containing another member of the tumour necrosis
factor receptors (LTBR) plus a TNF ligand (CD70) alongside the insulin receptor; and, (6) a small cluster containing Wnt-Receptors (FZD7, LRP6 and ROR2).
The PPI map was exported into CytoScape software (www.cytoscape.org) and the above-mentioned clusters were isolated (ClusterONE) for functional
analysis (BINGO). For each cluster, GO-annotations for biological processes were assigned and the number of involved proteins and corrected p-values were
calculated to assess enrichment of biological functions in the clusters compared to the entire human proteome (Supplementary Table S4 provides GO
annotations of all 6 identified clusters). Within cluster 1 the proteins are associated with cell surface receptor signalling (63%), integrin signalling (19%), cell
adhesion (50%), cell differentiation (44%) and cell migration (19%). Proteins in cluster 2 are involved in transmembrane receptor protein kinase signalling
(38%) and Ephrin signalling in particular (23%), as well as cell differentiation (46%), angiogenesis (15%) and, interestingly, neurogenesis (38%). Cluster 3
consists of various transporters responsible for organic acid transport (100%), amino acid transport (67%) and ion transport (50%) across the cell membrane.
The proteins in cluster 4 are involved in signal transduction again (67%), particularly in the insulin receptor pathway (33%). The HLA molecules in combination
with the IGF-1R play a role in the immune response (50%). Cluster 5 is involved in signal transduction (67%), cell migration (33%) and proliferation (33%).
Finally, cluster 6 is a small network of three receptors of the Wnt-signalling pathway and are thus involved in Wnt-signalling (100%), in tissue development,
particularly neurogenesis (67%) and cell proliferation (67%).
Additionally, to visualise cell surface localisation of these proteins, we performed an Ingenuity Pathway Analysis (Figure 2), which confirmed that the vast
majority of identified proteins are indeed cell surface molecules.
To verify the subcellular localisation and investigate functional networks in the set of highly upregulated surface proteins we built an IPA network of our
entries. IPA generates a network of 53 proteins and consecutively shows the subcellular localisation of the connected proteins. The vast majority (51) of these
53 proteins are indeed annotated to be localised in the plasma membrane, supporting our proteomic data. In the network, one protein is reported to be in the
extracellular space, and one, interestingly, is considered a nuclear protein. The remaining 70 proteins could not be connected to the generated network. Of the
remaining unconnected proteins, 5 were reported to be in the extracellular space, 4 in the cytoplasm and for 12 proteins the subcellular localisation was
unknown. The full list is provided in Supplementary Table S5, including subcellular localisation and molecular functions as annotated by IPA.
Figure 2: Ingenuity Pathway Analysis (IPA) of 123 significantly upregulated surface proteins on OS compared to healthy bone cells. The measure of
fold-change upregulation on OS cells compared to hp-OBs is indicated in a colour key; red for > 100-fold upregulation, dark pink for 41 to 99-fold and light pink
for 21 to 40-fold. Solid arrows represent direct interactions and dashed arrows represent indirect interactions between the proteins.