Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system

The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.     

Molecular cytogenetic analysis consistently identifies translocations involving chromosomes 1, 2 and 15 in five embryonal rhabdomyosarcoma cell lines and a PAX-FOXO1A fusion gene negative alveolar rhabdomyosarcoma cell line

Rhabdomyosarcoma in children is a "small round blue cell tumour" that displays skeletal muscle differentiation. Two main histological variants are recognised, alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma. Whereas consistent chromosome translocations characteristic of ARMS have been reported, no such cytogenetic abnormality has yet been described in ERMS. We have used multiple colour chromosome painting to obtain composite karyotypes for five ERMS cell lines and one PAX-FOXO1A fusion gene negative ARMS. The cell lines were assessed by spectral karyotyping (SKY), tailored multi-fluorophore fluorescence in situ hybridisation (M-FISH) using series of seven colour paint sets generated to examine specific abnormalities, and comparative genomic hybridisation (CGH). This approach enabled us to obtain karyotypes of the cell lines in greater detail than previously possible. Several recurring cytogenetic abnormalities were demonstrated, including translocations involving chromosomes 1 and 15 and chromosomes 2 and 15, in 4/6 and 2/6 cell lines respectively. All six cell lines demonstrated abnormalities of chromosome 15. Translocations between chromosomes 1 and 15 have previously been recorded in two primary cases of ERMS by conventional cytogenetics. Analysis of the translocation breakpoints may suggest mechanisms of ERMS tumourigenesis and may enable the development of novel approaches to the clinical management of this tumour.     

Sequential gel electrophoretic analysis of esterase-2 in two populations of Drosophila buzzatii

Sequential electrophoresis, using three different buffer systems on cellulose acetate gels, was used to characterize the allelic variation for esterase-2 in two populations of D. buzzatii in Australia that are separated by 550 km. Twenty-five alleles were detected, of which nine were unique to one population, eight unique to the other, and only eight were common to both populations. Allele frequencies within each population were significantly different between the two major chromosome sequences (standard and j inversion), and for each chromosome sequence allele frequencies were significantly different between populations. Observed allelic frequency distributions were not significantly different from those predicted for selective neutrality using the homozygosity test statistic. However, estimates of the effective sizes of the populations derived from their observed differentiation, together with the history of the species in Australia, provide support for some form of balancing selection affecting at least some of the alleles.     

Plasmid replication in Xenopus eggs and egg extracts: a 2D gel electrophoretic analysis.

We have examined the replication patterns of ribosomal DNA plasmids in vivo and in vitro using Xenopus eggs. Plasmids carrying different parts of the Xenopus ribosomal DNA sequence were allowed to replicate either in vitro in an egg extract or in vivo after microinjection into unfertilized eggs. The replication intermediates were analyzed by the 2D gel electrophoretic technique of Brewer and Fangman (1), using original or modified electrophoresis conditions. With standard electrophoresis conditions, the patterns obtained for restriction fragments larger than 5 kb were unreliable because of artefactually distorted Y arcs and unrecognizable bubble arcs. Interpretable patterns could nevertheless be obtained using suitably modified electrophoresis parameters. Under these conditions, replication was found to initiate and terminate at multiple, random locations on each plasmid both in vivo and in vitro. However, only one or very few of these potential initiation sites are used during the replication of an individual plasmid molecule. We discuss the possible artefacts and misinterpretations that can result when the 2D electrophoresis parameters are not adapted to the size of the fragment examined. We also discuss the relevance of the random replication mode to the mechanisms and the control of DNA replication in eukaryotes.     

Preparation and application of a partially degradable gel in mass spectrometry-based proteomic analysis

In-gel digestion is an attractive route in mass spectrometry-based proteomic analysis, which, however, often suffers from a certain amount of sample loss mainly due to insufficient protein digestion and peptide extraction. To address this, herein we establish a partially degradable gel-assisted protein digestion and peptide recovery method by means of a simple replacement of bis-acrylamide (BA) with bis-acrylylcystamine (BAC). Concretely, the protein sample solubilized using high concentrations of sodium dodecyl sulfate (SDS) and urea were directly entrapped and immobilized into BAC-crosslinked gel by vacuum-dried gel absorption followed by fixation treatment. After removal of SDS and urea by repeated washing, the proteins were subjected to in-gel digestion and the gel was reductively treated. The tryptic peptides were recovered from the partial degradation of the gel and analyzed afterwards by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). Compared with conventional BA-crosslinked gel method, this new method increased the numbers of identified proteins and unique peptides by 20.2% and 20.4%, respectively. The further statistical analysis demonstrated that the method improved the recovery of tryptic peptides particularly larger and/or hydrophobic peptides, thereby significantly facilitating protein identification. Thus, the newly developed method is a promising alternative for BA-crosslinked gel-based shotgun workflows and has potential application in the related fields of protein chemistry and proteomics.     

Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms

Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.     

H-2 antigens as genetic markers: a two-dimensional gel electrophoretic study

Direct immunoprecipitation and two-dimensional (2D) gel electrophoresis have been used to identify and characterize genetic variation of the H-2K and H-2D regions. Using inbred strains of mice and alloantisera, haplotype-specific polypeptides were defined for five different H-2 haplotypes. Specific immunoprecipitates prepared from strains of different haplotypes were applied to 2D gels in pairwise combinations to determine whether peptides specific to one haplotype can be distinguished from peptides specific to another. Those haplotype-specific peptides that migrate to unique positions on 2D gels with respect to the positions occupied by haplotype-specific peptides of another haplotype are useful as biochemical genetic markers. Cross-reactivity among K- and D-region antigens of different haplotypes was identified on 2D gels and found to correlate well with existing data based on serological cross-reactivity. An anti-mouse beta 2-microglobulin serum was found to be a useful general reagent for immunoprecipitating haplotype-specific H-2 antigens to permit their visualization on 2D gels.     

Differential serum proteomic analysis in a model of metabolic disease

Protein profiling would aid in better understanding the pathophysiology of metabolic disease. Here, we report on differential proteomic analysis using an animal model of diabetes mellitus and associated metabolic disorders (Otsuka Long-Evans Tokushima Fatty rat). Serum was analyzed by a new two-dimensional liquid chromatography system which separated proteins by chromatofocusing and subsequent reversed-phase chromatography. This is the first application of this approach to differential serum proteomics. Differentially expressed proteins, identified with MALDI-TOF mass spectrometry, included apolipoproteins and alpha2-HS-glycoprotein. These findings add to our understanding of the underlying pathophysiology. This new proteomic analysis is a promising tool to elucidate disease mechanisms.     

Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method

More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, B(act), and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, B(act), and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.     

Correlation analysis of two-dimensional gel electrophoretic protein patterns and biological variables

Background  

Two-dimensional gel electrophoresis (2DE) is a powerful technique to examine post-translational modifications of complexly modulated proteins. Currently, spot detection is a necessary step to assess relations between spots and biological variables. This often proves time consuming and difficult when working with non-perfect gels. We developed an analysis technique to measure correlation between 2DE images and biological variables on a pixel by pixel basis. After image alignment and normalization, the biological parameters and pixel values are replaced by their specific rank. These rank adjusted images and parameters are then put into a standard linear Pearson correlation and further tested for significance and variance.     

Modeling biological variability in 2-D gel proteomic carcinogenesis experiments

We propose a statistical method to model the underlying distribution of protein spot volumes in 2-D gels using a generalized model (GM). We apply this approach to discover mechanisms of chemical carcinogenesis in a rodent model. We generated 247 protein spots that were common to all gels (n = 18). Traditional statistical methods found 6.5% (13 out of 247) significant protein spots, our GM approach yielded a total of 53 (22.5%) differentially expressed protein spots.     

iTRAQ analysis of a cell culture model for malignant transformation, including comparison with 2D-PAGE and SILAC

To study human cancer development, cell culture models for malignant transformation can be used. In 1999 Hahn and Coworkers introduced such a model system and established herewith a basis for research on human tumorigenesis. Primary human fibroblasts are sequentially transduced with defined genetic elements (hTERT, SV40 ER, and H-RasV12), resulting in four defined cell lines, whereby the last has a fully transformed phenotype. In order to get a deeper insight into the molecular biology of human tumorigenesis, we compared the proteomes of these four cell lines following a multimethod concept. At the beginning we assumed SILAC and sample fractionation with COFRADIC is the method of choice to analyze the cell culture model for malignant transformation. Here, the compared samples are combined before sample preparation, thus avoiding differences in sample preparation, and using COFRADIC notably reduces sample complexity. Because 2D-PAGE is a standard method for the separation and visualization of closely related proteomes, we decided to analyze and compare the proteomes of these four cell lines in a first approach by differential 2D-PAGE. Surprisingly, we discovered much more unique results with iTRAQ and sample fractionation with SCX than with the combination of 2D-PAGE and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies not only in number of yielded results but also in analysis time. Here, we present the iTRAQ quantification results and compare them with the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the protein level at each transition. Thereby, SV40 has the strongest impact on the proteome. In detail we identified 201 regulated proteins. Beside others, these proteins are involved in cytoskeleton, RNA processing, and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins. For example, MCM proteins are up-regulated and collagens are down-regulated due to SV40 ER expression. Furthermore we made the observation that proteins containing the same domain have analogous regulation profiles during malignant transformation. For instance, several proteins containing a CH or LIM domain are down-regulated. Moreover, by this study and the defined cell culture model, changes could be clearly matched to specific steps during tumorigenesis.     

Development of a scale down cell culture model using multivariate analysis as a qualification tool

In characterizing a cell culture process to support regulatory activities such as process validation and Quality by Design, developing a representative scale down model for design space definition is of great importance. The manufacturing bioreactor should ideally reproduce bench scale performance with respect to all measurable parameters. However, due to intrinsic geometric differences between scales, process performance at manufacturing scale often varies from bench scale performance, typically exhibiting differences in parameters such as cell growth, protein productivity, and/or dissolved carbon dioxide concentration. Here, we describe a case study in which a bench scale cell culture process model is developed to mimic historical manufacturing scale performance for a late stage CHO‐based monoclonal antibody program. Using multivariate analysis (MVA) as primary data analysis tool in addition to traditional univariate analysis techniques to identify gaps between scales, process adjustments were implemented at bench scale resulting in an improved scale down cell culture process model. Finally we propose an approach for small scale model qualification including three main aspects: MVA, comparison of key physiological rates, and comparison of product quality attributes. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:152–160, 2014