Fig 1.
Schematic procedure for quantitative analysis of proteins in BC cells vs. normal bladder cells.
Fig 2.
Mass spectrometric analysis of stable isotope-labeled proteins (SILAC method).
(A) Determination of incorporation efficiency by MALDI-TOF/TOF-MS. Peaks annotated as R0 (left), R6 (middle), and R10 (right) are peptide GVVDSEDLPLNISR of heat shock protein 90 from HCV29, KK47, and YTS1 cells. (B) Identification and quantification of proteome in BC cells by 2D-HPLC LTQ Orbitrap MS. Peaks annotated as K0, K4, and K8 (left) and R0, R6, and R10 (right) are doubly charged peptide VNQIGSVTESLQACK of alpha enolase and GGPEVQQVPAGER of fatty acid synthase.
Fig 3.
Distributions of proteins identified in various experiments described in the text.
(A) Venn diagrams of numbers of identified proteins from individual experiments. (B) Ratios of KK47/HCV29 (M/L) and YTS1/HCV29 (H/L) for the set of 1766 proteins. log2 of the SILAC ratio for each protein (n = 2) reflects differences in relative expression among KK47, YTS1, and HCV29 cells. (C) Distribution of SILAC M/L ratios. (D) Distribution of SILAC H/L ratios. (E) Cluster graph ("heat map") generated by hierarchical clustering of significant regulated proteins after averaging z-scores using a 95% cutoff.
Table 1.
Protein number, log2 ratio mean±SD, and z-scores of SILAC-labeled proteins.
Table 2.
Upregulated proteins in BC cells with >95% confidencea.
Table 3.
Downregulated proteins in BC cells with >95% confidencea.
Fig 4.
Functional classification of identified proteins using SWISS-PROT database based on universal GO annotation terms.
Proteins shown were linked to at least one annotation term within the GO molecular function (A), biological process (B), and cellular component (C) categories.
Fig 5.
Functional network analysis of differentially regulated proteins with z-score cutoff 95% observed in different stages of BC cells using Ingenuity Pathways Analysis (IPA).
(A and B) Top network functions of DNA replication, molecular transport, cell growth, and cell proliferation for upregulated proteins. (C and D) Top network functions of cellular movement, immune cell trafficking, and lipid metabolism. Solid lines: direct known interactions. Dashed lines: suspected or indirect interactions. White: proteins known to be in the network but not identified in our study.
Fig 6.
Confirmation of SILAC-determined protein and RNA abundances.
(A) SILAC L:M:H average ratios for six selected proteins. (B) Western blotting analysis of selected proteins. Proteins were transferred to a PVDF membrane, probed with their primary antibodies, and incubated with HRP-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies. (C) Densitometric quantitation of the protein levels. The protein was normalized by the tublin, and then compared to HCV29, which were arbitrarily set at 1.0. (D) Gene expression for the proteins was analyzed by qRT-PCR. Relative expression in comparison to control samples was analyzed by the 2−ΔΔCt method and represented as Log2. Expression of genes above Log2(2) or below Log2(1/2) was significantly upregulated or downregulated, respectively.
Fig 7.
Differential expression revealed by cell staining.
HCV29, KK47, and YTS1 cells were cultured and stained with six antibodies directed to identified proteins (MAGEA4, THY1, IGF2BP1, VIM, CTNNB1, FN1) labeled with Cy3 as described in M&M. Images are shown of merge images of Cy3-conjugated antibodies and DAPI staining of nuclei (objective magnification 60×). Scale bars: 70 μm.