Comparison of statistical approaches for the analysis of proteome expression data of differentiating neural stem cells

Comparative proteomic studies often use statistical tests included in the software for the analysis of digitized images of two-dimensional electrophoresis gels. As these programs include only limited capabilities for statistical analysis, many studies do not further describe their statistical approach. To find potential differences produced by different data processing, we compared the results of (1) Student's t-test using a spreadsheet program, (2) the intrinsic algorithms implemented in the Phoretix 2D gel analysis software, and (3) the SAM algorithm originally developed for microarray analysis. We applied the algorithms to proteome data of undifferentiated neural stem cells versus in vitro differentiated neural stem cells. We found (1) 367 spots differentially expressed using Student's t-test, (2) 203 spots using the algorithms in Phoretix 2D, and (3) 119 spots using the algorithms in SAM, respectively, with an overlap of 42 spots detected by all three algorithms. Applying different statistical approaches on the same dataset resulted in divergent set of protein spots labeled as statistically "significant". Currently, there is no agreement on statistical data processing of 2DE datasets, but the statistical tests applied in 2DE studies should be documented. Tools for the statistical analysis of proteome data should be implemented and documented in the existing 2DE software.     

Proteome approaches to characterize seed storage proteins related to ditelocentric chromosomes in common wheat (Triticum aestivum L.)

Changes in protein composition of wheat endosperm proteome were investigated in 39 ditelocentric chromosome lines of common wheat (Triticum aestivum L.) cv. Chinese Spring. Two-dimensional gel electrophoresis followed by Coomassie Brilliant Blue staining has resolved a total of 105 protein spots in a gel. Quantitative image analysis of protein spots was performed by PDQuest. Variations in protein spots between the euploid and the 39 ditelocentric lines were evaluated by spot number, appearance, disappearance and intensity. A specific spot present in all gels was taken as an internal standard, and the intensity of all other spots was calculated as the ratio of the internal standard. Out of the 1755 major spots detected in 39 ditelocentric lines, 1372 (78%) spots were found variable in different spot parameters: 147 (11%) disappeared, 978 (71%) up-regulated and 247 (18%) down-regulated. Correlation studies in changes in protein intensities among 24 protein spots across the ditelocentric lines were performed. High correlations in changes of protein intensities were observed among the proteins encoded by genes located in the homoeologous arms. Locations of structural genes controlling 26 spots were identified in 10 chromosomal arms. Multiple regulators of the same protein located at various chromosomal arms were also noticed. Identification of structural genes for most of the proteins was found difficult due to multiple regulators encoding the same protein. Two novel subunits (1B(Z,) 1BDz), the structure of which are very similar to the high molecular weight glutenin subunit 12, were identified, and the chromosome arm locations of these subunits were assigned.     

Sources of Variability among Replicate Samples Separated by Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DE) offers high-resolution separation for intact proteins. However, variability in the appearance of spots can limit the ability to identify true differences between conditions. Variability can occur at a number of levels. Individual samples can differ because of biological variability. Technical variability can occur during protein extraction, processing, or storage. Another potential source of variability occurs during analysis of the gels and is not a result of any of the causes of variability named above. We performed a study designed to focus only on the variability caused by analysis. We separated three aliquots of rat left ventricle and analyzed differences in protein abundance on the replicate 2D gels. As the samples loaded on each gel were identical, differences in protein abundance are caused by variability in separation or interpretation of the gels. Protein spots were compared across gels by quantile values to determine differences. Fourteen percent of spots had a maximum difference in intensity of 0.4 quantile values or more between replicates. We then looked individually at the spots to determine the cause of differences between the measured intensities. Reasons for differences were: failure to identify a spot (59%), differences in spot boundaries (13%), difference in the peak height (6%), and a combination of these factors (21). This study demonstrates that spot identification and characterization make major contributions to variability seen with 2DE. Methods to highlight why measured protein spot abundance is different could reduce these errors.     

Improved comparative proteome analysis based on two-dimensional gel electrophoresis

The purpose of this study was to test the extent to which differences in spot intensity can be reliably recognized between two groups of two-dimensional electrophoresis gels (pH 4-7, visualized with ruthenium fluorescent stain) each loaded with different amounts of protein from rat brain (power analysis). Initial experiments yielded only unsatisfactory results: 546 spots were matched from two groups of 6 gels each loaded with 200 microg and 250 microg protein, respectively. Only 72 spots were higher (p<0.05), while 58 spots were significantly lower in the 250-microg group. The construction of new apparatuses that allowed the simultaneous processing of 24 gels throughout all steps between rehydration and staining procedure considerably lowered the between-gel variation. This resulted in the detection of significant differences in spot intensities in 77-90% of all matched spots on gel groups with a 25% difference in protein load. This applied both when protein from 24 biological replicates was loaded onto two groups of 12 gels and when two pooled tissue samples were each loaded onto 6 gels. At a difference of 50% in protein load, more than 90% of all spots differed significantly between two experimental groups.     

Utility of dry gel from two-dimensional electrophoresis for peptide mass fingerprinting analysis of silkworm proteins

We compared the use of wet and dry two-dimensional electrophoresis (2-DE) gels for in-gel tryptic digestion and subsequent analysis by mass spectrometry, first using bovine serum albumin (BSA) as a model protein and then using unknown proteins from an extract of the silkworm midgut. The gel was either dried at 80 degrees C or left wet. Upon analysis of BSA, there was little difference in peptide recovery from 2-DE or in mass spectrum between the dry and the wet gels. The midgut extract was resolved into more than 1,100 protein spots by 2-DE, and 40 of these spots were sampled for further analysis. For all of the 40 proteins, the results obtained from dry and wet gels were quite similar in mass spectra and protein identification, although the relative amounts of peptides from tryptic digestion ranged from 45 to 146%. Based on these results, we confirmed the utility of dry electrophoretic gels for proteomics of insect extracts.     

Proteomic analysis of rice leaf, stem and root tissues during growth course

Rice proteins were isolated from leaf, stem and root tissues, harvesting at 1, 2, 4, 8 and 10 weeks after budding. Each tissue of each age was separately pulverized in liquid nitrogen, and the resulted tissue powders were suspended in 10% TCA-acetone and followed by acetone suspension to precipitate at low temperature, which resulted in the tissue-specific and age-specific protein mixture. The protein mixtures were separated by 2-DE using polyacrylamide gels (26 x 20 cm). The protein spots were identified by N-terminal sequence analysis and by MALDI and LC-MS/MS analyses after in-gel tryptic digestion. From a total of 4532 spots, 676 unique proteins were identified, of which 80 proteins (12%) were observed in all three tissues: leaf, stem and root. In addition, 45 (7%) were common in leaf and stem, 57 (8%) in stem and root, and 10 (2%) proteins in root and leaf. Also 141 unique proteins (21%) were observed only for leaf, 96 (14%) for stem, and 247 (36%) for root tissue. Proteins playing a role for photosynthesis and energy production were most abundant in leaf and stem, and those for cell defense were rich in roots.     

Using multiplex-staining to study changes in the maize leaf phosphoproteome in response to mechanical wounding

Mechanical wounding of 2-week-old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation which can modulate protein activities, and transmit signals within cellular pathways and networks. We have used multiplex-staining of high-resolution 2D gels for protein (Sypro Ruby) and phosphorylation (Pro-Q Diamond) as a strategy for quantifying changes in the stoichiometry of phosphorylation after wounding for 270 protein spots. Rigorous statistical analysis of the time-index data allowed us to accept patterns of change in 125 of the spots as non-random, and these patterns were assigned to five clusters. A reliable identity was assigned to 21 selected proteins, most of which have been previously described as phospho-proteins. The results suggest that analysis of protein spots from high-resolution 2D gels by multiplex-staining for protein plus phosphorylation is a strategy that can be broadly useful for study of how the phospho-proteome responds to abiotic stress.     

The holm oak leaf proteome: analytical and biological variability in the protein expression level assessed by 2-DE and protein identification tandem mass spectrometry de novo sequencing and sequence similarity searching

As a first approach in establishing the holm oak leaf proteome, we have optimised a protocol for this plant and tissue which includes the following steps: trichloroacetic acid-acetone extraction, two-dimensional gel electrophoresis (2-DE) on pH 5 to 8 linear gradient immobilised pH gradient strips as the first dimension, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels as the second one. Proteins were detected by Coomassie staining. Gel images were recorded and digitalized, and the protein spots quantified by using a linear regression equation of protein quantity on spot volume obtained against standard proteins. Analytical variance was calculated for one-hundred protein spots from three replicate 2-DE gels of the same protein extract. Biological variance was determined for the same protein spots from independent tissue extracts corresponding to leaves from different trees, or the same tree at different orientations or sampling times during a day. Values of 26% for the analytical variance and 58.6% for the biological variance among independent trees were obtained. These values provide a quantified and statistical basis for the evaluation of protein expression changes in comparative proteomic investigations with this species. A representative set of the major proteins, covering the isoelectric point range of 5 to 8 and the relative molecular mass(r) range of 14 to 78 kDa, were subjected to liquid chromatography-tandem mass spectrometry analysis. Due to the absence of Quercus DNA or protein sequence databases, a method based on the procedure reported by Liska and Shevchenko including de novo sequencing and BLAST similarity searching against other plant species databases was used for protein identification. Out of 43 analysed spots, 35 were positively identified. The identified proteins mainly corresponded to enzymes involved in photosynthesis and energetic metabolism, with a significant number corresponding to RubisCO.     

Proteome analysis of rat hippocampal neurons by multiple large gel two-dimensional electrophoresis

The goal of the present study was to detect as many protein spots as possible in mammalian cells using two-dimensional gel electrophoresis (2-DE). For proteome analysis, it is of importance to reveal as many proteins as possible. A single standard 2-DE gel (pH 3-10, 18 cm x 20 cm, 13.5% gel) could detect 853 spots from proteins of cultured rat hippocampal neurons when visualized by silver staining. To increase the resolution of the separation and the number of detectable proteins by 2-DE, we utilized seven different narrow pH range immobilized pH gradients in the first dimension. In the second dimension, fourteen long SDS polyacrylamide gels were used: seven 7.5% gels for the separation of high molecular mass proteins (> or = 40 kDa) and seven 13.5% gels for the separation of low molecular mass proteins (< or = 40 kDa). Three hundred and sixty microg of proteins from cultured hippocampal neurons were loaded on to individual gels and visualized by silver staining. All 14 gel images were assembled into a 70 cm x 67 cm cybergel that contained 6677 protein spots, thereby indicating that the utilization of the present strategy led to a 783% increase in the number of detected spots in comparison to the standard procedure. Loading double the amount (720 microg) of proteins on to a 13.5% gel led to a 184% increase in the number of detected spots, thereby indicating that the present strategy has a potential to display more protein spots in the cybergels.     

Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.     

A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography

Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.     

Two-dimensional fluorescence difference gel electrophoresis analysis of spermatogenesis in the rat

The molecular mechanisms underlying normal and pathological spermatogenesis remain poorly understood. We compared protein concentrations in different germ cell types to identify those proteins specifically or preferentially expressed at each stage of rat spermatogenesis. Crude cytosolic protein extracts and reversed-phase HPLC prefractionated cytosolic extracts from spermatogonia, pachytene spermatocytes, and early spermatids were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). By comparing gels and carrying out statistical analyses, we were able to identify 1274 protein spots with relative abundances differing significantly between the three cell types. We found that 265 of these spots displaying highly differential expression (ratio > or = 2.5 between two cell types), identified by mass fingerprinting, corresponded to 123 nonredundant proteins. The proteins clustered into three clades, corresponding to mitotic, meiotic, and post-meiotic cell types. The differentially expressed proteins identified by 2-D DIGE were confirmed and validated by western blotting and immunohistochemistry, in the few cases in which antibodies were available. 2-D DIGE appears a relevant proteomics approach for studying rat germ cell differentiation, allowing the establishment of the precise expression profiles for a relatively large number of proteins during normal spermatogenesis.     

Hierarchical grid transformation for image warping in the analysis of two-dimensional electrophoresis gels

Two-dimensional electrophoresis is a widely used method for separating a large number of proteins from complex protein mixtures and for revealing differential patterns of protein expressions. In the computer-assisted proteome research, the comparison of protein separation profiles involves several heuristic steps, ranging from protein spot detection to matching of unknown spots. An important prerequisite for efficient protein spot matching is the image warping step, where the geometric relationship between the gel profiles is modeled on the basis of a given set of known corresponding spots, so-called landmarks, and the locations of unknown spots are predicted using the optimized model. Traditionally, polynomial functions together with least squares optimization has been used, even though this approach is known to be incapable of modeling all the complex distortions inherent in electrophoretic data. To satisfy the need of more flexible gel distortion correction, a hierarchical grid transformation method with stochastic optimization is presented. The method provides an adaptive multiresolution model between the gels, and good correction performance in the practical cross-validation tests suggests that automatic warping of gel images could be based on this approach. We believe that the proposed model also has significance in the ultimate comparison of corresponding protein spots since the matching process should benefit from the closeness of the true spot pairs.     

A proteomic approach to study pea (Pisum sativum) responses to powdery mildew (Erysiphe pisi)

As a global approach to gain a better understanding of the mechanisms involved in pea resistance to Erysiphe pisi, changes in the leaf proteome of two pea genotypes differing in their resistance phenotype were analyzed by a combination of 2-DE and MALDI-TOF/TOF MS. Leaf proteins from control non-inoculated and inoculated susceptible (Messire) and resistant (JI2480) plants were resolved by 2-DE, with IEF in the 5-8 pH range and SDS-PAGE on 12% gels. CBB-stained gels revealed the existence of quantitative and qualitative differences between extracts from: (i) non-inoculated leaves of both genotypes (77 spots); (ii) inoculated and non-inoculated Messire leaves (19 spots); and (iii) inoculated and non-inoculated JI2480 leaves (12 spots). Some of the differential spots have been identified, after MALDI-TOF/TOF analysis and database searching, as proteins belonging to several functional categories, including photosynthesis and carbon metabolism, energy production, stress and defense, protein synthesis and degradation and signal transduction. Results are discussed in terms of constitutive and induced elements involved in pea resistance against Erysiphe pisi.     

Application of partial least squares discriminant analysis to two-dimensional difference gel studies in expression proteomics

Two-dimensional difference gel electrophoresis (DIGE) is a tool for measuring changes in protein expression between samples involving pre-electrophoretic labeling ith cyanine dyes. In multi-gel experiments, univariate statistical tests have been used to identify differential expression between sample types by looking for significant changes in spot volume. Multivariate statistical tests, which look for correlated changes between sample types, provide an alternate approach for identifying spots with differential expression. Partial least squares-discriminant analysis (PLS-DA), a multivariate statistical approach, was combined with an iterative threshold process to identify which protein spots had the greatest contribution to the model, and compared to univariate test for three datasets. This included one dataset where no biological difference was expected. The novel multivariate approach, detailed here, represents a method to complement the univariate approach in identification of differentially expressed protein spots. This new approach has the advantages of reduced risk of false-positives and the identification of spots that are significantly altered in terms of correlated expression rather than absolute expression values.     

Improvement of plant protein solubilization and 2-DE gel resolution through optimization of the concentration of Tris in the solubilization buffer

It is important to solubilize acetone-precipitated proteins before isoelectric focusing (IEF) to achieve high resolution 2-DE gels. To resolve the maximum possible number of plant protein spots, we developed an improved solubilization buffer for plant proteins. We demonstrated that the resolution of 2-DE gels increased dramatically as the concentration of Tris-base increased, with maximum solubilization obtained at 200 mM Tris-base (Ly200T). The Ly200T buffer was more effective than the commonly used solubilization buffer containing 40 mM Tris at solubilizing acetone-precipitated plant proteins. Use of the Ly200T buffer to solubilize proteins resulted in an increase in intensity of approximately 30% of plant protein spots in the larger-than-40 kDa region of the gel. The Ly200T buffer also improved the resolution of abundant and basic proteins. Thus, the Ly200T buffer can be used to achieve greater resolution of protein spots in plant proteomics research.     

Protein Changes in Response to Pyrene Stress in Maize （Zea mays L.） Leaves

Phytoremedlation is a relatively new approach to remove polycyclic aromatic hydrocarbons （PAHs） from the environment. When plants are grown under pyrene treatment, they respond by synthesizing a set of protective proteins. To learn more about protein changes in response to pyrene treatment, we extracted total proteins from the leaves of maize （Zea mays L.） 1 week after pyrene treatment. The proteins extracted were separated with twodimensional gel electrophoresis. In total, approximately 54 protein spots were found by comparing gels from treated and control groups. According to the Isoelectric point, molecular weight, and abundance of these protein spots, 20 pyrene-lnduced proteins were found to have changed abundance. Of these, 15 protein spots were Increased and five protein spots were newly appeared in pyrene-treated plant leaves. Six model upregulated protein spots of different molecular weights were excised from the gels and subjected to trypsin digestion followed by peptide separation using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Peptlde masses were used to search the matrix-science database for protein Identification. Two of the proteins were Identified on the basis of the homology of their peptide profiles with existing protein sequences as pyruvate orthophosphate diklnase and the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunlt. These proteins are Involved in the regulation of carbohydrate and energy metabolism. The present study gives new Insights into the pyrene stress response In maize leaves and demonstrates the power of the proteomlc approach in phytoremedlation of PAHs.     

Predicting failure rate of PCR in large genomes

We have developed statistical models for estimating the failure rate of polymerase chain reaction (PCR) primers using 236 primer sequence-related factors. The model involved 1314 primer pairs and is based on more than 80 000 PCR experiments. We found that the most important factor in determining PCR failure is the number of predicted primer-binding sites in the genomic DNA. We also compared different ways of defining primer-binding sites (fixed length word versus thermodynamic model; exact match versus matches including 1–2 mismatches). We found that the most efficient prediction of PCR failure rates can be achieved using a combination of four factors (number of primer-binding sites counted in different ways plus GC% of the primer) combined into single statistical model GM1. According to our estimations from experimental data, the GM1 model can reduce the average failure rate of PCR primers nearly 3-fold (from 17% to 6%). The GM1 model can easily be implemented in software to premask genome sequences for potentially failing PCR primers, thus improving large-scale PCR-primer design.     

Proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis (2D-DIGE)

Salt stress is a major abiotic stress that limits agricultural productivity in many regions of the world. To understand the molecular basis of the salt stress response in wheat (Triticum aestivum L.), a proteomic approach was used to identify the salt stress-responsive proteins in an elite Chinese wheat cultivar, Zhengmai 9023, which exhibits a high yield, superior gluten quality and better biotic resistance. Three-week-old seedlings were treated with NaCl of four different concentrations (1.0%, 1.5%, 2.0%, and 2.5%). The total proteins from the leaves of untreated and NaCl-treated plants were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2358 protein spots were detected on the gels, among which 125 spots showed a significant change in protein abundance, and 83 differentially expressed spots were localised on preparative gels. Using Q-TOF mass spectrometry, 52 salt-responsive spots were identified, which were classified into six functional categories that included transport-associated proteins, detoxifying enzymes, ATP synthase, carbon metabolism, protein folding, and proteins with unknown biological functions. Of the 52 differentially expressed proteins, 26 were up-regulated, 21 were down-regulated, and five spots showed multi-expression patterns. In particular, some important proteins for salt tolerance were found to be up-regulated in Zhengmai 9023 under salt stress, such as H⁺-ATPases, glutathione S-transferase, ferritin and triosephosphate isomerase.     

Protein extraction from the earthworm Eisenia fetida for 2‐DE

We identified an efficient protocol for extracting proteins from whole earthworm, Eisenia fetida, for 2‐DE. Sample preparation is a critical step in a 2‐DE proteome approach and is absolutely essential for obtaining good results. Six protein extraction protocols based on different protein precipitation agents were tested and evaluated using 2‐DE. The methods generated remarkably different 2‐DE protein spot patterns. We conclude that trichloroacetic acid (TCA)‐A eliminates interfering compounds, thus allowing for the efficient resolubilization of proteins. TCA‐A gives good distinction, more bands in 1‐DE gels, and the most number of protein spots in 2‐DE gels. It is also rapid, provides the higher protein yield, and has the less number of steps. To demonstrate the quality of the extracted proteins, we cut several protein spots that were common to four methods from 2‐DE gels, analyzed them using MALDI‐TOF/TOF MS, and tentatively identified them. The classic TCA‐A method proved to be most useful as a standard method of extracting proteins from E. fetida.