Springer Science and Business Media LLC

Three dimensional (3D) growths of cancer cells in vitro are more reflective of in situ cancer cell growth than growth in monolayer (2D). The present study is designed to determine changes in protein and phosphoprotein that reflect adaptation of tumor cells to 3D as compared to 2D. Since relative hypoxia is a common feature of most solid tumors, the present study also aims to look at the impact of transition from normoxia to hypoxia in these two growth conditions. Using reverse-phase protein arrays, we compared levels of 121 different phosphorylated and non-phosphorylated proteins in 5 glioma and 6 adenocarcinoma lines under conditions of 3D and monolayer culture in normoxia and hypoxia. A three-way analysis of variance showed levels of 82 antibodies differed between media (2D vs. 3D) and 49 differed between treatments (hypoxia vs. normoxia). Comparing 2D to 3D growth, 7 proteins were commonly (i.e., > 50% of tumors) elevated in 3D: FAK, AKT, Src, GSK3αβ, TSC2, p38, and NFκβp65. Conversely, 7 other proteins are commonly decreased: ATRIP, ATR, β-catenin, BCL-X, cyclin B1, Egr-1, and HIF-1α. Comparing normoxia to hypoxia, only NCKIPSD was commonly elevated in hypoxia; 6 proteins were decreased: cyclin B1, 4EBP1(Ser65), c-Myc, SMAD3(Ser423), S6(Ser235), and S6(Ser240). Hypoxia affected glioma cell lines differently from adenocarcinoma cell lines: 8 proteins were increased in gliomas (BAX, caspase 7, HIF-1α, c-JUN, MEK1, PARP 1 cleaved, Src, and VEGFR2) and none in adenocarcinomas. We identified subsets of proteins with clearly concordant/discordant behavior between gliomas and adenocarcinomas. In general, monolayer to 3D culture differences are clearer than normoxia to hypoxia differences, with anti-apoptotic, cytoskeletal rearrangement and cell survival pathways emphasized in the former and mTOR pathway, transcription, cell-cycle arrest modulation, and increased cell motility in the latter.

Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

The silkworm Bombyx mori is a lepidopteran insect with four developmental stages: egg, larva (caterpillar), pupa, and adult. The hemolymph of the silkworm is in an open system that circulates among all organs, and functions in nutrient and hormone transport, injury, and immunity. To understand the intricate developmental mechanisms of metamorphosis, silkworm hemolymph from different developmental stages, including the 3rd day of fifth instar, the 6th day of fifth instar, the 3rd day of pupation, the 8th day of pupal stage and the first day of the moth stage, was investigated by two-dimensional electrophoresis and mass spectrometry. Two-dimensional polyacrylamide gel electrophoresis showed that from the larval to moth stages, silkworm hemolymph proteins changed markedly. Not only did major proteins such as SP1, SP2, and the 30 K lipoprotein change, but other proteins varied greatly at different stages. To understand the functions of these proteins in silkworm development, 56 spots were excised from gels for analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 34 proteins involved in metamorphosis, programmed cell death, food digestion, metabolism, and nutrient storage and transport. Most proteins showed different expression at different stages, suggesting functions in development and metamorphosis. An abundance of proteins related to immunity were found, including hemolin, prophenoloxidase, serine proteinase-like protein, paralytic peptide-binding protein, and protease inhibitor. Proteomics research not only provides the opportunity for direct investigation of protein expression patterns, but also identifies many attractive candidates for further study. Two-dimensional maps of hemolymph proteins expressed during the growth and metamorphosis of the silkworm offer important insights into hemolymph function and insect metamorphosis.

Numerous gel-based softwares exist to detect protein changes potentially associated with disease. The data, however, are abundant with technical and structural complexities, making statistical analysis a difficult task. A particularly important topic is how the various softwares handle missing data. To date, no one has extensively studied the impact that interpolating missing data has on subsequent analysis of protein spots. This work highlights the existing algorithms for handling missing data in two-dimensional gel analysis and performs a thorough comparison of the various algorithms and statistical tests on simulated and real datasets. For imputation methods, the best results in terms of root mean squared error are obtained using the least squares method of imputation along with the expectation maximization (EM) algorithm approach to estimate missing values with an array covariance structure. The bootstrapped versions of the statistical tests offer the most liberal option for determining protein spot significance while the generalized family wise error rate (gFWER) should be considered for controlling the multiple testing error. In summary, we advocate for a three-step statistical analysis of two-dimensional gel electrophoresis (2-DE) data with a data imputation step, choice of statistical test, and lastly an error control method in light of multiple testing. When determining the choice of statistical test, it is worth considering whether the protein spots will be subjected to mass spectrometry. If this is the case a more liberal test such as the percentile-based bootstrap t can be employed. For error control in electrophoresis experiments, we advocate that gFWER be controlled for multiple testing rather than the false discovery rate.

In contrast to blood, which has mechanisms to maintain a homeostatic internal environment, urine is more likely to reflect changes in the body. As urine accumulates all types of changes, identifying the precise cause of changes in the urine proteome is challenging and crucial in biomarker discovery. To reduce the effects of both genetic and environmental factors on the urinary proteome, this study used a rat model of adriamycin-induced nephropathy resembling human focal segmental glomerulosclerosis (FSGS) development. Urine samples were collected at before adriamycin administration and day3, 7, 11, 15 and 23 after. Urinary proteins were profiled by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Of 23 changed proteins with disease development, 20 have human orthologs, and 13 proteins were identified as stable in normal human urine, meaning that changes in these proteins are more likely to reflect disease. Fifteen of the identified proteins have not been established to function in FSGS development. Seven proteins were selected for verification in ten more rats as markers closely associated with disease severity by western blot. We identified proteins changed in different stages of FSGS in rat models, which may aid in biomarker development and the understanding of FSGS pathogenesis.

TGF-β acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-β can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-β in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01. Preliminary experiments based on two-dimensional electrophoresis of a hydrophobic cell fraction identified only 5 differentially expressed proteins from BRI-JM01 cells. Since 3 of these proteins were glycoproteins, we next used the lectin, wheat germ agglutinin (WGA), to enrich for glycoproteins, followed by relative quantification of tryptic peptides using a label-free LC-MS based method. Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin). Interestingly, despite the fact that TGF-β induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap. These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments. Alternatively, it is possible that the two cell lines may use different mechanisms to achieve an EMT transition.

Helicobacter hepaticus colonizes the intestine and liver of mice causing hepatobiliary disorders such as hepatitis and hepatocellular carcinoma, and has also been associated with inflammatory bowel disease in children. In its habitat, H. hepaticus must encounter bile which has potent antibacterial properties. To elucidate virulence and host-specific adaptation mechanisms of H. hepaticus modulated by human or porcine bile, a proteomic study of its response to the two types of bile was performed employing two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The 2-DE and mass spectrometry analyses of the proteome revealed that 46 proteins of H. hepaticus were differentially expressed in human bile, 18 up-regulated and 28 down-regulated. In the case of porcine bile, 32 proteins were differentially expressed of which 19 were up-regulated, and 13 were down-regulated. Functional classifications revealed that identified proteins participated in various biological functions including stress response, energy metabolism, membrane stability, motility, virulence and colonization. Selected genes were analyzed by RT-PCR to provide internal validation for the proteomic data as well as provide insight into specific expressions of motility, colonization and virulence genes of H. hepaticus in response to human or porcine bile. Overall, the data suggested that bile is an important factor that determines virulence, host adaptation, localization and colonization of specific niches within host environment.

In this study a proteomic approach was used to investigate the steady-state response of Escherichia coli to temperature up-shifts in a cascade of two continuously operated bioreactors. The first reactor served as cell source with optimal settings for microbial growth, while in the second chemostat the cells were exposed to elevated temperatures. By using this reactor configuration, which has not been reported to be used for the study of bacterial stress responses so far, it is possible to study temperature stress under well-defined, steady-state conditions. Specifically the effect on the cellular adaption to temperature stress using two-dimensional gel electrophoresis was examined and compared at the cultivation temperatures of 37°C and 47.5°C. As expected, the steady-state study with the double bioreactor configuration delivered a different protein spectrum compared to that obtained with standard batch experiments in shaking flasks and bioreactors. Setting a high cut-out spot-to-spot size ratio of 5, proteins involved in defence against oxygen stress, functional cell envelope proteins, chaperones and proteins involved in protein biosynthesis, the energy metabolism and the amino acid biosynthesis were found to be differently expressed at high cultivation temperatures. The results demonstrate the complexity of the stress response in a steady-state culture not reported elsewhere to date.