Identification of Missing Proteins in the Phosphoproteome of Kidney Cancer

Identifying missing proteins (MPs) has been one of the critical missions of the Chromosome-Centric Human Proteome Project (C-HPP). Since 2012, over 30 research teams from 17 countries have been trying to search adequate and accurate evidence of MPs through various biochemical strategies. MPs mainly fall into the following classes: (1) low-molecular-weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), (4) nucleic acid-associated proteins, (5) low abundance, and (6) unexpressed genes. In this study, kidney cancer and adjacent tissues were used for phosphoproteomics research, and 8962 proteins were identified, including 6415 phosphoproteins, and 44 728 phosphosites, of which 10 266 were unreported previously. In total, 75 candidate detections were found, including 45 phoshoproteins. GO analysis for these 75 candidate detections revealed that these proteins mainly clustered as membrane proteins and took part in nephron and kidney development. After rigorous screening and manual check, 9 of them were verified with the synthesized peptides. Finally, only one missing protein was confirmed. All mass spectrometry data from this study have been deposited in the PRIDE with identifier PXD006482

Identification of Missing Proteins in the Phosphoproteome of Kidney Cancer

Identifying missing proteins (MPs) has been one of the critical missions of the Chromosome-Centric Human Proteome Project (C-HPP). Since 2012, over 30 research teams from 17 countries have been trying to search adequate and accurate evidence of MPs through various biochemical strategies. MPs mainly fall into the following classes: (1) low-molecular-weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), (4) nucleic acid-associated proteins, (5) low abundance, and (6) unexpressed genes. In this study, kidney cancer and adjacent tissues were used for phosphoproteomics research, and 8962 proteins were identified, including 6415 phosphoproteins, and 44 728 phosphosites, of which 10 266 were unreported previously. In total, 75 candidate detections were found, including 45 phoshoproteins. GO analysis for these 75 candidate detections revealed that these proteins mainly clustered as membrane proteins and took part in nephron and kidney development. After rigorous screening and manual check, 9 of them were verified with the synthesized peptides. Finally, only one missing protein was confirmed. All mass spectrometry data from this study have been deposited in the PRIDE with identifier PXD006482

Notch1 and Jagged1 mRNA expression analysis at different stages in ccRCC samples.

<p>(A) Analysis showing significantly higher expression of Notch1 in metastatic tumors at T1 stage compared to localized tumors(P = 0.001). (B) No statistically significant difference of Jagged1 expression in all three stages. The data shown are mean±SD.</p

Analysis of cell cycle with over-expression of Notch1 and Jagged1.

<p>(A)–(B) In 786-O, Caki-1 and HKC cell line, cell cycle analysis demonstrating no difference between cells over-expressing Notch1 and Jagged1 compared to controls. (C) P21 and P27 remained unchanged in all cell lines after transfected with Notch1 and Jagged1 plasmids.</p

Notch1 promoting the proliferation in tumor and normal kidney cell lines.

<p>Proliferation assay by MTS showing increased proliferation in Caki-1 and 786-O cell line after Notch1 and Jagged1 expression. MTS assay in HKC cell line showing increased proliferation rate in Notch1 expressing cells only. The data shown are means±SD from two independent experiments, each carried out in triplicate.</p

Analysis of tumor diameter and Notch1 and Jagged1 mRNA expression level in T1 stage ccRCC.

<p>(A) Analysis showing larger average diameter in metastatic tumors (6.375±0.479 cm n = 4) compared to localized tumors (4.089±1.237 cm, n = 19, P = 0.025). (B) Positive correlation of Notch1 expression and tumor diameter in T1 stage(n = 23, R = 0.435, P = 0.038). (C) No correlation of Jagged1 expression and tumor size in T1 stage(n = 23, R = −0.172, P = 0.432). The data shown are mean±SD.</p

Notch1 and Jagged1 promoting the migration in tumor cell lines.

<p>(A)–(C) are representative view of 786-O cell line transfected with Notch1, Jagged1 and control plasmid respectively. (D)–(F) are Caki-1 cell line transfected with Notch1, Jagged1 and control plasmid respectively. (G) Notch1 and Jagged1 can promote the migration of 786-O and Caki-1 tumor cell lines compared to the controls. (H) MMP-9 mRNA expression was significantly elevated in Caki-1 and 786-O cell lines after transfected with Notch1 and Jagged1 plasmid. The data shown are mean±SD.</p

Notch1 and Jagged1 expression in ccRCC tissues.

<p>(A) mRNA expression detected by real-time RT-PCR showing elevated Notch1 mRNA expression in localized and metastatic tumors compared to non-tumor(NT) tissues (P = 0.001 and P = 0.000 respectively), and higher expression in metastatic tumors compared to localized tumors (P = 0.028); On the right panel, analysis showing elevated Jagged1 mRNA expression in tumors compared to NT (P = 0.000 and P = 0.000 for localized and metastatic tumors respectively), but lower expression in metastatic tumors compared to localized tumors (P = 0.005). Each dot representing a tissue sample. (B) Protein expression detected by western-blot assay showing elevated expression of Notch1 and Jagged1 protein in tumor tissues compared to non-tumor tissues. The right panel was the densitometric analysis of the bands. The data shown are mean±SD.</p

Effects of E2F1 in migration and invasion of cancer cells.

<p>(A) Representative view of 786-O migration and invasion transfected with E2F1 plasmid, entry plasmid and nothing blank respectively. (B) Representative view of A498 migration and invasion transfected with E2F1 plasmid, entry plasmid and nothing blank respectively. (C) Representative view of Caki-1 migration and invasion transfected with E2F1 siRNA sequence and negative control. (D) MMP2, MMP9, vimentin and ZEB1 were validated in three groups as aggressiveness related genes. MMP2, MMP9 mRNA expressions in E2F1 group were significantly elevated compared with untransfected and entry groups in 786-O and A498 cells. The data shown were mean±SD. Each Experiment was done in triplicate.</p

<b>ccRCC</b> tissues(38 cases) and their corresponding adjacent non-cancerous kidney tissues were immunohistochemically stained by E2F1 antibody(1:100).

<p>Three representative photographs were taken at different magnifications in ccRCC tissues(A-B: 200× A, 400× B and negative control of tumor cells of C) and corresponding adjacent non-cancerous kidney tissues(D-E:200× D,400×E and negative control of normal renal tubules cells of F) respectively.</p