Bile samples from 15 CC and 10 cholangitis patients (JI 101 site pooled as described in 
the Material and Methods) over a pH range of 3?0 (Figure 1). Analysis of the 2-DE gels revealed 109 spots were differently expressed in the pooled CC and cholangitis bile samples (P,0.05). In total, 97 spots corresponding to 49 genes were successfully identified via MALDI-TOF/TOF. Additionally, a number of proteins yielded more than two spots in the profiles (Table S1). Among the 97 proteins, 61 proteins were present in a higherProteomic Study Reveals SSP411 as a CC Biomarkerabundance in bile from CC patients compared to the cholangitis group. Bioinformatic analysis of the 49genes by the BioGPS database (http://biogps.org/#goto = welcome) revealed that eight genes were uniquely expressed by the liver, while the other 14 were highly expressed in the liver or fetal liver (Figure S1). These results suggest that the successfully identified differentially expressed proteins were derived from bile.Verification of candidate biomarkers in the pooled and individual bile samplesTo verify the proteomic analysis, five proteins were randomly selected for immunoblotting analysis: PGAM-1, PDIA3, HSPD1 and SSP411 which were upregulated in CC bile and APOM which was downregulated in CC bile. The proteins migrated at the expected molecular weights (Figure 1) and Western blotting revealed that the expression levels of these proteins in the pooled CC and cholangitis bile samples were essentially consistent with the 2D-PAGE results (Figure 2). Additionally, Western blotting of SSP411, PGAM-1, PDIA3 and HSPD1 in individual bile samples provided identical results to the pooled bile samples. In Western blots of crude bile (2 ml), PGAM1 and SSP411 were barely detectable in the benign group but were expressed at high levels in the CC group (Figure 3A and D). PDIA3 and HSPD1 (Figure 3B and C) could be Apocynin custom synthesis detected in several cholelithiasis patients; however, the average expression levels of PDIA3 and HSPD1 in crude bile from CC patients was significantly higher.from CC patients) in eight pairs of CC and adjacent non-tumor bile duct tissues. As shown in Figure 4, PGAM-1, HSPD1, PDIA3 and SSP411 were overexpressed in tumor tissues compared to the matched non-tumor tissues. Immunohistochemical analysis was performed to characterize the distribution of PGAM-1, HSPD1, PDIA3 and SSP411 in surgical tumor tissues. All four proteins were upregulated in CC (Figure 5, right panel) compared to the non-tumor tissues came from patients with choledocholithiasis (Figure 5, left panel). Intense PGAM-1, HSPD1, PDIA3 and SSP411 cytoplasmic immunoreactivity was observed in both hilar cholangiocarcinoma (Figure 5) and intrahepatic cholangiocarcinoma (Figure S2). The lumen of the cancer nests also demonstrated various intensities of immunostaining, suggesting that the proteins can be secreted extracellularly. The immunohistochemical analysis provided further confirmation that PGAM-1, HSPD1, PDIA3 and SSP411 were overexpressed in CC, suggesting that these proteins may provide potential biomarkers for CC.Evaluation of the serum levels of SSP411 by ELISA as a diagnostic testAs bile can only be collected during surgery, bile 1313429 biomarkers are not suitable as a pre-surgical diagnostic tool. Therefore, we examined the serum levels of one potential biomarker. BioGPS analysis indicated SSP411 is a testis-enriched gene which is not expressed in normal liver (Figure S1C). SSP411 has not previously been associated with other c.Bile samples from 15 CC and 10 cholangitis patients (pooled as described in the Material and Methods) over a pH range of 3?0 (Figure 1). Analysis of the 2-DE gels revealed 109 spots were differently expressed in the pooled CC and cholangitis bile samples (P,0.05). In total, 97 spots corresponding to 49 genes were successfully identified via MALDI-TOF/TOF. Additionally, a number of proteins yielded more than two spots in the profiles (Table S1). Among the 97 proteins, 61 proteins were present in a higherProteomic Study Reveals SSP411 as a CC Biomarkerabundance in bile from CC patients compared to the cholangitis group. Bioinformatic analysis of the 49genes by the BioGPS database (http://biogps.org/#goto = welcome) revealed that eight genes were uniquely expressed by the liver, while the other 14 were highly expressed in the liver or fetal liver (Figure S1). These results suggest that the successfully identified differentially expressed proteins were derived from bile.Verification of candidate biomarkers in the pooled and individual bile samplesTo verify the proteomic analysis, five proteins were randomly selected for immunoblotting analysis: PGAM-1, PDIA3, HSPD1 and SSP411 which were upregulated in CC bile and APOM which was downregulated in CC bile. The proteins migrated at the expected molecular weights (Figure 1) and Western blotting revealed that the expression levels of these proteins in the pooled CC and cholangitis bile samples were essentially consistent with the 2D-PAGE results (Figure 2). Additionally, Western blotting of SSP411, PGAM-1, PDIA3 and HSPD1 in individual bile samples provided identical results to the pooled bile samples. In Western blots of crude bile (2 ml), PGAM1 and SSP411 were barely detectable in the benign group but were expressed at high levels in the CC group (Figure 3A and D). PDIA3 and HSPD1 (Figure 3B and C) could be detected in several cholelithiasis patients; however, the average expression levels of PDIA3 and HSPD1 in crude bile from CC patients was significantly 
higher.from CC patients) in eight pairs of CC and adjacent non-tumor bile duct tissues. As shown in Figure 4, PGAM-1, HSPD1, PDIA3 and SSP411 were overexpressed in tumor tissues compared to the matched non-tumor tissues. Immunohistochemical analysis was performed to characterize the distribution of PGAM-1, HSPD1, PDIA3 and SSP411 in surgical tumor tissues. All four proteins were upregulated in CC (Figure 5, right panel) compared to the non-tumor tissues came from patients with choledocholithiasis (Figure 5, left panel). Intense PGAM-1, HSPD1, PDIA3 and SSP411 cytoplasmic immunoreactivity was observed in both hilar cholangiocarcinoma (Figure 5) and intrahepatic cholangiocarcinoma (Figure S2). The lumen of the cancer nests also demonstrated various intensities of immunostaining, suggesting that the proteins can be secreted extracellularly. The immunohistochemical analysis provided further confirmation that PGAM-1, HSPD1, PDIA3 and SSP411 were overexpressed in CC, suggesting that these proteins may provide potential biomarkers for CC.Evaluation of the serum levels of SSP411 by ELISA as a diagnostic testAs bile can only be collected during surgery, bile 1313429 biomarkers are not suitable as a pre-surgical diagnostic tool. Therefore, we examined the serum levels of one potential biomarker. BioGPS analysis indicated SSP411 is a testis-enriched gene which is not expressed in normal liver (Figure S1C). SSP411 has not previously been associated with other c.