Comparative analysis of platelet depleted plasma prepared on the Roche 8100 automation line and manually centrifuged platelet poor plasma for routine coagulation assays.

Objectives: To evaluate whether the routine coagulation tests can be performed using platelet depleted plasma (PDP, residual platelet count <40000/µL) to achieve maximum efficiency of the automated workflow and compare results of these tests performed with platelet poor plasma (PPP residual platelet count <10,000/µL) prepared manually 'offline'. Design and Methods: The PDP was obtained first following 'on line' centrifugation at 4150 RPM (3000g) for 7 min. The routine coagulation tests, Prothrombin Time (PT), Activated Partial Thromboplastin Clotting Time (aPTT), D-dimer (DD), Antithrombin III (AT3) and Fibrinogen (FBG) were performed. The PPP was obtained from an aliquot of PDP samples with additional 'manual off line' centrifugation at 7700 RPM (3314g) for 3 min (total 10 min, online + offline) and the same tests were performed. The statistical analysis was carried out using EP Evaluator v11 to compare results from both methods. Results: The results from both PPP and PDP samples demonstrated strong correlation. For example, PT (R = 0.9989; N = 55, and of Bias -0.12 (-0.67%), aPTT(R = 0.9957; N = 60, Bias 0.26 (0.58%)), AT3(R = 0.9800; N = 49, Bias -2.0 (-2.2%)), FBG (R = 0.9956; N = 57, Bias -1.9 (-0.5%)) and DD (R = 0.9981; N = 38, Bias 0.005 (0.373%)) with insignificant bias. Conclusions: The utilization of the Roche cobas® 8100 automated 'online' centrifugation helps achieve optimal workflow efficiency without impacting analytical performance of the PT, aPTT, DD, AT3 and FBG assays. The use of PDP can be superior method to PPP for routine coagulation tests.

Analytical and Clinical Performance of Two Point of Care Rapid Antibody Assays for SARS-CoV-2.

OBJECTIVE: The SARS-CoV-2 pandemic has reached to a state where populations across the world should ad-just to live with it like many other diseases. Regular serosurveys are essential for disease surveil-lance and policy decisions. In this study, we evaluated the analytical and clinical performance of two commercially available rapid antibody assays. METHODS: SARS-CoV-2 PCR positive patients (N=104) were recruited for method evaluation study of two commercially available lateral flow Rapid IgM and IgG assays; Edinburgh Genetics ActivXpress+ COVID-19 IgG/IgM Immunoassay Complete Testing Kit (EGCV0092L) and Abchek COVID-19 IgM/IgG Antibody Rapid Test (NUL/COV-19/R&D/001). We have tested all the participants for SARS-CoV-2 with a Rapid Anti-gen Test (Abchek) on the day of sample collection. Additionally, we analyzed vaccinated people (N=187) for seroprevalence of IgG. EP Evaluator version 12 and GraphPad Prism 9.5.0 were used for statistical analysis. RESULTS: The IgG seropositivity after 10-15 days of PCR positivity was 97.11% (Edinburg Genetics Assay) and 92.30% (Abchek Assay). The Rapid Antigen test was 100% negative with IgM negativity of 93.27% (Edinburg Genetics Assay) and 98.08% (Abchek Assay). The IgG seropositivity of vaccinated participants was 89.84% using both the assays. The IgG sero-positivity was 86.82% (Edinburg Genetics Assay, N=91) and 92.71% (Abchek Assay, N=96) in the study participants with post vaccination. CONCLUSIONS: These assays are robust and scalable. Both the assays can be used for serosurveys with desired scale and speed when a quick observation is needed for surveillance. These tests are cost effective, field deployable without need of any sophis-ticated instruments and large capital. IMPACT STATEMENT: During a public health emergency like the COVID-19 pandemic, regular sero-surveys are essential for disease surveillance and swift policy decisions. However, deployment of the gold standard methods like quantitative ELISA, neutralizing antibody assays for assessment of population based seroprevalence and immune status becomes logistically difficult, costly and time consuming. The point of care antibody assays are easily scalable, affordable and field deployable. The present study demonstrates that these tests are reliable in terms of analytical and clinical performance. These assays could be used when rapid observations are to be made by including large sample population.

Comparison of Performance between the Elecsys 4th and 5th Generation STAT Cardiac Troponin T Immunoassays.

OBJECTIVE: To evaluate the analytical and clinical performance of the 5th GEN cTnT assay by comparing it with the 4th GEN assay. METHODS: Imprecision, analytical measurement range (AMR), reference interval, and quantitative comparison were studied. Qualitative comparisons of the two assays for randomly selected patients with the cTnT test orders and patients with elevated N-terminal proB-type natriuretic peptide (NT-proBNP), decreased estimated glomerular filtration rate (eGFR), and increased procalcitonin were performed. RESULTS: Within-run and between-run CVs of the 5th GEN assay were 0.6-2.3% and 3.1-5.0% with AMR of 8-10,000 ng/L and reference interval of <19 ng/L. Regression analysis of the two assays showed linear relationship: y (5th GEN)=0.899x (4th GEN)+26, r=0.9993 with a positive bias of the 5th GEN assay in samples with cTnT <150 ng/L and a negative bias in samples with cTnT >500 ng/L compare to the 4th GEN assay. Agreement was 88.1% (95% CI: 80.4 to 93.1%) between the two methods according to the 99th percentile threshold. In patients with elevated NT-proBNP, decreased eGFR, and increased procalcitonin, agreements were 79.4%, 92.7%, and 100%. CONCLUSION: The 5th GEN cTnT assay demonstrates excellent precision and acceptable AMR. Compared to the 4th GEN assay, the 5th GEN assay shows a negative bias for samples with cTnT >500 ng/L but a positive bias for samples with cTnT <150 ng/L and identifies more patients with elevated cTnT.

Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.

Gallbladder cancer (GBC) is an aggressive gastrointestinal malignancy with no approved targeted therapy. Here, we analyze exomes (n = 160), transcriptomes (n = 115), and low pass whole genomes (n = 146) from 167 gallbladder cancers (GBCs) from patients in Korea, India and Chile. In addition, we also sequence samples from 39 GBC high-risk patients and detect evidence of early cancer-related genomic lesions. Among the several significantly mutated genes not previously linked to GBC are ETS domain genes ELF3 and EHF, CTNNB1, APC, NSD1, KAT8, STK11 and NFE2L2. A majority of ELF3 alterations are frame-shift mutations that result in several cancer-specific neoantigens that activate T-cells indicating that they are cancer vaccine candidates. In addition, we identify recurrent alterations in KEAP1/NFE2L2 and WNT pathway in GBC. Taken together, these define multiple targetable therapeutic interventions opportunities for GBC treatment and management.

Automated Measurement of Plasma Cell-Free Hemoglobin Using the Hemolysis Index Check Function.

BACKGROUND: The Roche Cobas chemistry analyzer's hemolysis index (HI) check function can directly report hemoglobin (Hb) concentrations. We aimed to validate the HI check function for the measurement of plasma cell-free Hb. METHODS: Plasma samples (6 µl) were taken by the analyzer and diluted in normal saline to measure the absorbance for Hb at 570 and 600 nm. Hb concentrations were calculated based on the molar extinction coefficient. Imprecision, lower limit of quantification (LLOQ), and analytical measurement range (AMR) of the assay were evaluated. The accuracy was determined by comparing the results between the new method and an existing spectrophotometric method. We further studied interference of icterus and lipemia and carryover. The performance of the assay in proficiency testing was also evaluated. The reference range was transferred from the existing method. RESULTS: Within-run and total CVs were 1.7%-4.2% and 2.1%-7.0%, respectively (n = 20). The LLOQ was 11 mg/dL (CV = 8.1%) with the upper limit of AMR of 506 mg/dL. The results of the new method correlated well with the existing reference assay: Y (new method) = 0.974 x (reference method) + 4.9, r = 0.9990, n = 52. Bilirubin with a concentration up to 60 mg/dL and lipemic index up to 389 did not show significant interference. No significant carryover was detected. The average standard deviation index in proficiency testing was 0.03 ± 0.29. The reference range was <22 mg/dL. CONCLUSIONS: Plasma cell-free Hb measurement using the HI check function meets the analytical requirements of the plasma cell-free Hb assays. It is simple and cost-effective.

MYC and Twist1 cooperate to drive metastasis by eliciting crosstalk between cancer and innate immunity.

Metastasis is a major cause of cancer mortality. We generated an autochthonous transgenic mouse model whereby conditional expression of MYC and Twist1 enables hepatocellular carcinoma (HCC) to metastasize in >90% of mice. MYC and Twist1 cooperate and their sustained expression is required to elicit a transcriptional program associated with the activation of innate immunity, through secretion of a cytokinome that elicits recruitment and polarization of tumor associated macrophages (TAMs). Systemic treatment with Ccl2 and Il13 induced MYC-HCCs to metastasize; whereas, blockade of Ccl2 and Il13 abrogated MYC/Twist1-HCC metastasis. Further, in 33 human cancers (n = 9502) MYC and TWIST1 predict poor survival (p=4.3×10-10), CCL2/IL13 expression (p<10-109) and TAM infiltration (p<10-96). Finally, in the plasma of patients with HCC (n = 25) but not cirrhosis (n = 10), CCL2 and IL13 were increased and IL13 predicted invasive tumors. Therefore, MYC and TWIST1 generally appear to cooperate in human cancer to elicit a cytokinome that enables metastasis through crosstalk between cancer and immune microenvironment.

PIM1 kinase promotes gallbladder cancer cell proliferation via inhibition of proline-rich Akt substrate of 40 kDa (PRAS40).

Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC.

O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis.

Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked ß-N-acetylglucosamine (O-GlcNAcylation) posttranslational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs.

Identification of spleen tyrosine kinase as a potential therapeutic target for esophageal squamous cell carcinoma using reverse phase protein arrays.

The vast majority of esophageal cancers in China, India and Iran are esophageal squamous cell carcinomas (ESCC). A timely diagnosis provides surgical removal as the main therapeutic option for patients with ESCC. Currently, there are no targeted therapies available for ESCC. We carried out reverse phase protein array-based protein expression profiling of seven ESCC-derivedcell lines and a non-neoplastic esophageal epithelial cell line (Het-1A) to identify differentially expressed proteins in ESCC. SYK non-receptortyrosine kinase was overexpressed in six out of seven ESCC cell lines that were used in the study. We evaluated the role of SYK in ESCC using the pharmacological inhibitor entospletinib (GS-9973) and siRNA-based knock down studies. Entospletinib is a selective inhibitor of SYK, which is currently being evaluated in phase II clinical trials for hematological malignancies. Using in vivo subcutaneous tumor xenografts in mice, we demonstrate that treatment with entospletinib significantly inhibits tumor growth. Further clinical studies are needed to prove the efficacy of entospletinib as a targeted therapeutic agent for treating ESCC.

Discovery of noncanonical translation initiation sites through mass spectrometric analysis of protein N termini.

Translation initiation generally occurs at AUG codons in eukaryotes, although it has been shown that non-AUG or noncanonical translation initiation can also occur. However, the evidence for noncanonical translation initiation sites (TISs) is largely indirect and based on ribosome profiling (Ribo-seq) studies. Here, using a strategy specifically designed to enrich N termini of proteins, we demonstrate that many human proteins are translated at noncanonical TISs. The large majority of TISs that mapped to 5' untranslated regions were noncanonical and led to N-terminal extension of annotated proteins or translation of upstream small open reading frames (uORF). It has been controversial whether the amino acid corresponding to the start codon is incorporated at the TIS or methionine is still incorporated. We found that methionine was incorporated at almost all noncanonical TISs identified in this study. Comparison of the TISs determined through mass spectrometry with ribosome profiling data revealed that about two-thirds of the novel annotations were indeed supported by the available ribosome profiling data. Sequence conservation across species and a higher abundance of noncanonical TISs than canonical ones in some cases suggests that the noncanonical TISs can have biological functions. Overall, this study provides evidence of protein translation initiation at noncanonical TISs and argues that further studies are required for elucidation of functional implications of such noncanonical translation initiation.

Therapeutic potential of an anti-angiogenic multimodal biomimetic peptide in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. Due to inadequate screening methods and the common coexistence of limited functional liver reserves, curative treatment options are limited. Liver transplantation is the only curative treatment modality for early HCC. There are multidisciplinary treatment options like ablative treatments, radiation and systemic therapy available for more advanced patients or those that are inoperable. Treatment resistance and progression is inevitable for these HCC patients. Newer therapeutics need to be explored for better management of HCC. HCC is a hypervascular tumor and many pro-angiogenic proteins are found significantly overexpressed in HCC. Here we explored the therapeutic potential of the anti-angiogenic, anti-lymphangiogenic, and directly anti-tumorigenic biomimetic collagen IV-derived peptide developed by our group. Human HCC cell lines HuH7, Hep3b and HepG2 showed significant disruption of cell adhesion and migration upon treatment with the peptide. Consistent with previously described multimodal inhibitory properties, the peptide was found to inhibit both c-Met and IGF1R signaling in HepG2 cells and blocked HepG2 conditioned media stimulation of microvascular endothelial cell (MEC) tube formation. Furthermore, the peptide treatment of mouse HepG2 tumor xenografts significantly inhibited growth relative to untreated controls. The peptide was also found to improve the survival of autochthonous Myc-induced HCC in a transgenic mouse model. Mechanistically, we found that the peptide treatment reduced microvascular density in the autochthonous liver tumors with increased apoptosis. This study shows the promising therapeutic potential of our biomimetic peptide in the treatment of HCC.

Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer.

Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors.In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies.In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05).The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%).Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.

The non-receptor tyrosine kinase TNK2/ACK1 is a novel therapeutic target in triple negative breast cancer.

Breast cancer is the most prevalent cancer in women worldwide. About 15-20% of all breast cancers do not express estrogen receptor, progesterone receptor or HER2 receptor and hence are collectively classified as triple negative breast cancer (TNBC). These tumors are often relatively aggressive when compared to other types of breast cancer, and this issue is compounded by the lack of effective targeted therapy. In our previous phosphoproteomic profiling effort, we identified the non-receptor tyrosine kinase TNK2 as activated in a majority of aggressive TNBC cell lines. In the current study, we show that high expression of TNK2 in breast cancer cell lines correlates with high proliferation, invasion and colony forming ability. We demonstrate that knockdown of TNK2 expression can substantially suppress the invasiveness and proliferation advantage of TNBC cells in vitro and tumor formation in xenograft mouse models. Moreover, inhibition of TNK2 with small molecule inhibitor (R)-9bMS significantly compromised TNBC proliferation.Finally, we find that high levels of TNK2 expression in high-grade basal-like breast cancers correlates significantly with poorer patient outcome. Taken together, our study suggests that TNK2 is a novel potential therapeutic target for the treatment of TNBC.

Macrophage migration inhibitory factor - a therapeutic target in gallbladder cancer.

BACKGROUND: Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer. METHODS: Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA. RESULTS: The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells. CONCLUSIONS: Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.

Global phosphotyrosine survey in triple-negative breast cancer reveals activation of multiple tyrosine kinase signaling pathways.

Breast cancer is the most prevalent cancer in women worldwide. About 15-20% of all breast cancers are triple negative breast cancer (TNBC) and are often highly aggressive when compared to other subtypes of breast cancers. To better characterize the biology that underlies the TNBC phenotype, we profiled the phosphotyrosine proteome of a panel of twenty-six TNBC cell lines using quantitative high resolution Fourier transform mass spectrometry. A heterogeneous pattern of tyrosine kinase activation was observed based on 1,789 tyrosine-phosphorylated peptides identified from 969 proteins. One of the tyrosine kinases, AXL, was found to be activated in a majority of aggressive TNBC cell lines and was accompanied by a higher level of AXL expression. High levels of AXL expression are correlated with a significant decrease in patient survival. Treatment of cells bearing activated AXL with a humanized AXL antibody inhibited cell proliferation and migration in vitro, and tumor growth in mice. Overall, our global phosphoproteomic analysis provided new insights into the heterogeneity in the activation status of tyrosine kinase pathways in TNBCs. Our approach presents an effective means of identifying important novel biomarkers and targets for therapy such as AXL in TNBC.

Calcium calmodulin dependent kinase kinase 2 - a novel therapeutic target for gastric adenocarcinoma.

Gastric cancer is one of the most common gastrointestinal malignancies and is associated with poor prognosis. Exploring alterations in the proteomic landscape of gastric cancer is likely to provide potential biomarkers for early detection and molecules for targeted therapeutic intervention. Using iTRAQ-based quantitative proteomic analysis, we identified 22 proteins that were overexpressed and 17 proteins that were downregulated in gastric tumor tissues as compared to the adjacent normal tissue. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was found to be 7-fold overexpressed in gastric tumor tissues. Immunohistochemical labeling of tumor tissue microarrays for validation of CAMKK2 overexpression revealed that it was indeed overexpressed in 94% (92 of 98) of gastric cancer cases. Silencing of CAMKK2 using siRNA significantly reduced cell proliferation, colony formation and invasion of gastric cancer cells. Our results demonstrate that CAMKK2 signals in gastric cancer through AMPK activation and suggest that CAMKK2 could be a novel therapeutic target in gastric cancer.

Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous-cell carcinoma.

Esophageal squamous-cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early-stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non-neoplastic Het-1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry-based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA-based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation.

Identification of prosaposin and transgelin as potential biomarkers for gallbladder cancer using quantitative proteomics.

Gallbladder cancer is an uncommon but lethal malignancy with particularly high incidence in Chile, India, Japan and China. There is a paucity of unbiased large-scale studies investigating molecular basis of gallbladder cancer. To systematically identify differentially regulated proteins in gallbladder cancer, iTRAQ-based quantitative proteomics of gallbladder cancer was carried out using Fourier transform high resolution mass spectrometry. Of the 2575 proteins identified, proteins upregulated in gallbladder cancer included several lysosomal proteins such as prosaposin, cathepsin Z and cathepsin H. Downregulated proteins included serine protease HTRA1 and transgelin, which have been reported to be downregulated in several other cancers. Novel biomarker candidates including prosaposin and transgelin were validated to be upregulated and downregulated, respectively, in gallbladder cancer using tissue microarrays. Our study provides the first large scale proteomic characterization of gallbladder cancer which will serve as a resource for future discovery of biomarkers for gallbladder cancer.