Proteomic capacity of recent fluorescent dyes for protein staining

Chevalier, François; Rofidal, Valérie; Váňová, Pavlína; Bergoin, Alexis; Rossignol, Michel

doi:10.1016/j.phytochem.2004.04.019

Cited by 81 publications

(85 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The LDR for two proteins, one of high and the other of low abundance with relatively similar mobility, was linear (15.6 ng-4 μg) with a correlation value of 0.99. Resolution of this native extract by 2DE resulted in 250 detectable spots, the lowest total spot number obtained in comparison to all stains tested in the study [17].…”

Section: Coomassie Brilliant Bluementioning

confidence: 72%

“…The Neuhoff formulation of cCBB was applied to a 2D separation of a total soluble protein extract derived from Arabidopsis thaliana to determine detection sensitivity [17]. Here, it was shown that the LLD for the total protein extract, consisting of a complex mixture of proteins with varying mobilities and abundance, was 2 μg of the protein loaded.…”

Section: Coomassie Brilliant Bluementioning

confidence: 99%

“…While comparable between silver and SR stained protein at higher loads, MS coverage for two standard proteins (PhosB and OVA) was decreased at lower protein loads for silver stain (without glutaraldehyde). A silver nitrate staining method developed by Mortz et al [74] was applied to A. thaliana total soluble protein extract separated by 1D and 2D PAGE [17]. The LLD was determined to be 1 ng (i.e., more sensitive than cCBB), but showed lower correlation for two unspecified protein bands arbitrarily selected by the authors (band 1, R=0.82; band 2, R=0.88).…”

Section: Silver Stainmentioning

confidence: 99%

“…With these techniques, protein resolution has been substantially and routinely improved. Taken together, all of these developments indicate that the main limitation to the amount of proteomic information obtained from 2DE is unlikely to be its capacity to resolve proteins but rather to be one of protein detection (i.e., stain sensitivity) [17].…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

View full text Add to dashboard Cite

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

show abstract

Section: Coomassie Brilliant Bluementioning

confidence: 72%

Section: Coomassie Brilliant Bluementioning

confidence: 99%

Section: Silver Stainmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

View full text Add to dashboard Cite

show abstract

“…The resulting spot patterns can be visualized by labeling the sample prior to the separation, e.g. with radioactivity (21) or fluorescence (1,6,22), or after the separation with total protein stains such as colloidal Coomassie (2), SYPRO Ruby, or Deep Purple (23,24). The spot volumes can be compared from one sample to another, and it is an established technique in expression studies.…”

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

View full text Add to dashboard Cite

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...

show abstract

Proteomics

Casado¹,

Casal²

2010

Pharmaceutical Sciences Encyclopedia

View full text Add to dashboard Cite

Proteomics is a branch of study dedicated to the biological implication of proteins in disease, pathologies, or alterations of living organisms. One of the most important steps in a proteomic study is the selection of the biological material to work with. The article describes various methods and steps in a proteomic study, including electrospray ionization of proteins and multidimensional liquid chromatography.

show abstract

Proteomic capacity of recent fluorescent dyes for protein staining

Cited by 81 publications

References 23 publications

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

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

Proteomics

Contact Info

Product

Resources

About