Data analysis methods for detection of differential protein expression in two-dimensional gel electrophoresis

Meunier, Bruno; Bouley, J.; Piec, Isabelle; Bernard, Carine; Picard, Brigitte; Hocquette, Jean-François

doi:10.1016/j.ab.2005.02.028

Cited by 67 publications

(61 citation statements)

References 18 publications

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“…Although there have been numerous statistical analyses performed with DIGE investigations, there is presently no consensus strategy for identifying differential proteins. 31,34,35 We present here an alternative that is simple yet robust for minimizing false-positives.…”

Section: A Threementioning

confidence: 99%

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Proteomic Analysis of Near-Isogenic Sunflower Varieties Differing in Seed Oil Traits

et al. 2007

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Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mass were comparatively analyzed in biological triplicate at 18 days after flowering using two-dimensional (both pI 3-10 and 4-7) Difference Gel Electrophoresis. Additionally, two inbred lines varying in oleic acid content, HA89 (18% oleic) and HA341 (89% oleic), were also analyzed in the same manner. Statistical analyses of these sunflower lines was performed beginning with fitting a mixed effects linear model to the log-transformed optical volume of each spot to account for gel variation, followed by testing the significance between varieties for mean transformed optical spot volumes. The p-values from the spot analysis procedures were then used to find the cutoff point for differential expression using a 10% false-discovery rate (FDR). Comparison of the oil content and oleic acid composition lines revealed 77 and 42 protein spots below the 10% FDR cutoff, respectively, and were therefore declared differentially expressed. Liquid chromatography-tandem mass spectrometry analysis of each of these protein spots resulted in assignments for 44 and 17 spots, respectively. Fructokinase, plastid phosphoglycerate kinase, and enolase proteins were determined to be up-regulated in the high oil line, while phosphofructokinase, cytosolic phosphoglucomutase, and cytsolic phosphoglycerate kinase were up-regulated in the low oil variety. Additionally, four activities involved in amino acid synthesis were up-regulated in the low oil variety in addition to 12S storage proteins and a protein similar to legumin storage protein. Interestingly, two 2-DE spots identified as 14-3-3 proteins were found to be up-regulated in high oleic acid variety. Alteration of glycolytic and amino acid biosynthetic enzymes, as well as storage protein levels, suggests seed oil content is tightly linked to carbohydrate metabolism and protein synthesis in a complex manner.

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Section: A Threementioning

confidence: 99%

“…FDR is used extensively in microarray transcript profiling experimentation and has also been used by a few groups to characterize proteome data sets. 9,31,34,35 In this paper, we applied FDR for selection of deferentially expressed spots. FDR is a statistical procedure that is effective in correcting for multiple simultaneous hypothesis tests.…”

Section: A Threementioning

confidence: 99%

Proteomic Analysis of Near-Isogenic Sunflower Varieties Differing in Seed Oil Traits

et al. 2007

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“…Later, the image of the polyacrylamide gel was digitized in ImageScanner™ of GE Healthcare (compatible with ImageMaster software) and then analyzed by the program Image Master Platinum. The protein bands were quantified in volume units (area vs. intensity) following the method described by Meunier et al (2005).…”

Section: Detoxification Of Castor Bean and Quantification Of Ricinmentioning

confidence: 99%

Use of castor meal (Ricinus communis L.) as a source of dietary protein in goats during the mating period: impact on reproductive and metabolic responses

et al. 2015

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Use of castor meal (Ricinus communisL AbstractThe aim of the present study was to evaluate the effects of the total substitution of soybean meal with castor meal, detoxified or non-detoxified, on the response to estrous synchronization, conception rate, early fetal development, presence of IgG, and metabolic-hormonal response. Sixty mixed goats were fed diets without castor meal (WCM), with detoxified castor meal (DCM), and with castor meal (CM) during early pregnancy. The goats had their estrous synchronized and were then submitted to the mating season. The number of fetuses was determined by ultrasonography after 25 days of mating and their development was followed until 60 days of gestation. Plasma levels of progesterone (P 4 ), liver enzymes, and urea were determined along with the evaluation of the immunological response. After 15 days of experimental feeding, immunoglobulin G (IgG) was detected by western blotting only in goats that received non-detoxified castor meal. There was no effect (p > 0.05) of type of diet on response to estrous synchronization, plasma P 4 levels, conception rate, or embryonic/fetal development. In pregnant goats, there was an effect of diet (p < 0.001) on plasma urea levels in multiple-birth pregnancy, gammaglutamyltransferase (GGT) in single-birth pregnancy, and lactate dehydrogenase (LDH) in both types of pregnancy. In non-pregnant goats, there were increased urea levels in all types of diets and in LDH in WCM goats, but GGT levels decreased in the WCM and CM goats when compared with pregnant goats (p > 0.05). In addition, plasma levels of LDH in WCM goats and of urea in all types of diet were higher in non-pregnant goats than pregnant goats. In conclusion, it can be inferred that the inclusion of 15% castor meal, whether or not it is detoxified, to the diet of goats does not affect the reproductive performance, embryonic and early fetal development, or blood metabolites. ResumoO objetivo do presente estudo foi avaliar o efeito da substituição total do farelo de soja por farelo de mamona detoxificado ou não sobre a resposta à sincronização do estro, taxa de concepção, desenvolvimento fetal inicial, presença de IgG e resposta metabólica-hormonal. Sessenta cabras mestiças foram alimentadas sem farelo de mamona (SFM), com farelo de mamona detoxificado (FMD) e com farelo de mamona (FM) durante a gestação inicial. Todos os animais tiveram o estro sincronizado e depois foram acasaladas por monta natural. A partir do 25º dia após a monta, foi determinado o número de fetos e realizado o acompanhado do desenvolvimento dos mesmos por ultrassonografia até os 60 dias de gestação. Foi avaliado o perfil de progesterona (P 4 ), os níveis de metabólitos e a resposta imunológica. A partir do 15º dia de alimentação a imunoglobulina G (IgG) foi marcada através da técnica de Western Blotting, apenas em animais que receberam farelo de mamona não detoxificado. Não houve efeito (p>0,05) do tipo de dieta sobre a resposta à sincronização do estro, níveis plasmáticos de P 4 , taxa de gestação e desenvo...

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“…In multivariate methods, such as principle component analysis, all the data are simultaneously analyzed to look for patterns in expression. Each of these statistical methods involves a number of mathematical assumptions; however, these assumptions are often ignored, potentially leading to erroneous results (8). Regardless of the analytical method used to collect the proteomics data, subtle issues with statistical analysis must be overcome to ensure that valid conclusions are made.…”

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confidence: 99%

“…To date, the majority of published quantitative proteomics studies have used the established 2D gel electrophoresis approach for quantitation and applied a univariate test to identify protein species with significant changes in expression (8), although an increased number of studies are now using non-gel-based approaches. These tests calculate the probability (p) of observing a test statistic more extreme than the one calculated from the data given that the samples are from the same population (i.e.…”

mentioning

confidence: 99%

Experimental and Statistical Considerations to Avoid False Conclusions in Proteomics Studies Using Differential In-gel Electrophoresis

Karp

McCormick²,

Russell

et al. 2007

Molecular & Cellular Proteomics

152

137

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In quantitative proteomics, the false discovery rate (FDR) can be defined as the number of false positives within statistically significant changes in expression. False positives accumulate during the simultaneous testing of expression changes across hundreds or thousands of protein or peptide species when univariate tests such as the Student's t test are used. Currently most researchers rely solely on the estimation of p values and a significance threshold, but this approach may result in false positives because it does not account for the multiple testing effect. For each species, a measure of significance in terms of the FDR can be calculated, producing individual q values. The q value maintains power by allowing the investigator to achieve an acceptable level of true or false positives within the calls of significance. The q value approach relies on the use of the correct statistical test for the experimental design. In this situation, a uniform p value frequency distribution when there are no differences in expression between two samples should be obtained. Here we report a bias in p value distribution in the case of a three-dye DIGE experiment where no changes in expression are occurring. The bias was shown to arise from correlation in the data from the use of a common internal standard. With a two-dye schema, where each sample has its own internal standard, such bias was removed, enabling the application of the q value to two different proteomics studies. In the case of the first study, we demonstrate that 80% of calls of significance by the more traditional method are false positives. In the second, we show that calculating the q value gives the user control over the FDR. These studies demonstrate the power and ease of use of the q value in correcting for multiple testing. This work also highlights the need for robust experimental design that includes the appropriate application of statistical procedures. Molecular & Cellular Proteomics 6: 1354 -1364, 2007.Quantitative proteomics, the study of global changes in protein expression, is a rapidly growing field where two-dimensional (2D) 1 gel electrophoresis (1, 2), differential labeling of protein and peptides with stable isotopes (3), and label-free mass spectrometric peak intensity measurements (4) are pivotal approaches to measuring changes in the expression level of proteins. The output of large scale genomic sequencing and gene expression studies have driven the need to globally assess protein behavior, which is central to our understanding of cellular function and disease processes. In quantitative studies, the quantitation of the protein or peptide signal allows the comparison of protein levels from one state with another. Regardless of the technique used, the data generated can be analyzed with a variety of statistical approaches. In a simple comparison of two samples, changes in protein expression are considered to be significant when above a specified threshold (5). More rigorous studies incorporating replicates use univariate (1, 6) or multivari...

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Data analysis methods for detection of differential protein expression in two-dimensional gel electrophoresis

Cited by 67 publications

References 18 publications

Proteomic Analysis of Near-Isogenic Sunflower Varieties Differing in Seed Oil Traits

Proteomic Analysis of Near-Isogenic Sunflower Varieties Differing in Seed Oil Traits

Use of castor meal (Ricinus communis L.) as a source of dietary protein in goats during the mating period: impact on reproductive and metabolic responses

Experimental and Statistical Considerations to Avoid False Conclusions in Proteomics Studies Using Differential In-gel Electrophoresis

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