ABSTRACT
Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen(®), and AMMP(®)) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253.
Subject(s)
Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Liver Cirrhosis/genetics , Liver Neoplasms/genetics , Neoplasm Proteins/genetics , Phosphoproteins/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Aged , Aged, 80 and over , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Calnexin/genetics , Calnexin/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Dipeptidases/genetics , Dipeptidases/metabolism , Endoplasmic Reticulum Chaperone BiP , Female , Gene Expression Profiling , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Liver/metabolism , Liver/pathology , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Middle Aged , Molecular Sequence Annotation , Neoplasm Proteins/metabolism , Phosphoproteins/metabolism , Phosphorylation , Procollagen-Proline Dioxygenase/genetics , Procollagen-Proline Dioxygenase/metabolism , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , Proteomics/methods , Signal Transduction , Thioredoxins/genetics , Thioredoxins/metabolism , Valosin Containing ProteinABSTRACT
The acoustic membrane micro particle (AMMP) technology has been used to quantify single analytes out of multiple sample types. In this study the technology is used to reveal molecular interactions of components of kinase pathways. Specifically, the downstream kinase activity of the EGFR receptor in the presence or absence of EGFR inhibitors is investigated. These experiments substantiate that EGFR stimulation predominantly activates the MEK/ERK pathway. The EGFR inhibitors tested had varying effectiveness at preventing phosphorylation at the EGFR or downstream kinase activity. These experiments reveal the use of the AMMP technology for observing multiple signaling pathways in a single experiment.
Subject(s)
ErbB Receptors/metabolism , Protein Kinase Inhibitors/pharmacology , Cell Line, Tumor , ErbB Receptors/antagonists & inhibitors , Humans , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/metabolism , MCF-7 Cells , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Quinolines/chemistry , Quinolines/pharmacology , Signal TransductionABSTRACT
Effective drug discovery demands the availability of microgram to gram quantities of high-quality protein encoded by novel transcripts. Protein expression vectors designed for large-scale protein production often include one or more specific tags to such transcripts, to simplify the purification of the targeted protein. Optimization of the complex expression and purification process requires the evaluation of multiple expression candidate clones to identify a production-suitable construct in terms of quality and final protein yield. Efficiency of the entire expression screening process is typically assessed by direct visualization of the banding patterns from whole-cell lysates on SDS-PAGE gels, by direct staining and/or immunoblotting, using antibodies against the tag or the protein of interest. These techniques, generally run under denaturing conditions, have proven to be only marginally predictive of the purification yield and authentic folding for native proteins. Small-scale, multiparallel affinity purification followed by SDS-PAGE analysis is more predictive for expression screening; however, this approach is labor intensive and time consuming. Here we describe the development of an alternative expression efficiency assessment technique, designed to evaluate the accessibility of affinity tags expressed with the desired fusion proteins, using acoustic membrane microparticle assay technology on the ViBE protein analysis workstation.
Subject(s)
Immunoassay/methods , Recombinant Fusion Proteins/analysis , Animals , Cloning, Molecular , Escherichia coli/genetics , Histidine , Indicators and Reagents/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Sf9 Cells , SpodopteraABSTRACT
Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays. An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass. To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate. Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.
