Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches

Righetti, Pier Giorgio; Castagna, Annalisa; Antonucci, Francesca; Piubelli, Chiara; Cecconi, Daniela; Campostrini, Natascia; Antonioli, Paolo; Astner, Hubert; Hamdan, Mahmoud

doi:10.1016/j.chroma.2004.05.106

Cited by 95 publications

(56 citation statements)

References 34 publications

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“…Cys residues are also less likely to be at trypsin cleavage sites. As such, proteins labeled at cys residues can still be identified by MS following standard digestion protocols [133,146,147]. Focusing on thiols also allows the pIs of labeled proteins to be maintained since the dyes are neutrally charged.…”

Section: Reactive Fluorescent Dyesmentioning

confidence: 99%

“…In particular, labeling lysine (lys) residues facilitates near complete coverage of a proteome given the prevalence of lys in proteins [132]. This may, however, affect the efficiency of subsequent trypsin treatment if the reactive dye masks the lys residues [132,133]; nonetheless, there are a range of alternative reagents available for the controlled digestion of proteins to defined peptides, as is required prior to MS analysis [134][135][136].…”

Section: Reactive Fluorescent Dyesmentioning

confidence: 99%

“…Each of these programs also offers a differing array of options for editing images, statistical analysis and user interaction depending on the users' needs. A comparison of different analytical programs rated PDQuest (Bio-Rad) as best able to deal with spot overlap but described Melanie III (Genebio) and Progenesis SameSpots (Nonlinear Dynamics) as "all-rounders" in terms of accuracy and coping with low S/N ratios [133]. A comparison of Delta2D and Proteomweaver (Bio-Rad)…”

Section: Equipment Innovationsmentioning

confidence: 99%

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

2010

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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.

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Section: Reactive Fluorescent Dyesmentioning

confidence: 99%

Section: Reactive Fluorescent Dyesmentioning

confidence: 99%

Section: Equipment Innovationsmentioning

confidence: 99%

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

2010

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“…Molecular analysis of RNA has rapidly evolved from classical techniques such as the Northern blot to current large scale, high throughput microarray analyses (1)(2)(3)(4). Proteomics offers an alternate means of expression analysis (5)(6)(7)(8)(9). As a direct rather than surrogate measure of protein expression, proteomics has evolved, like genomics, toward the capacity for routine, reliable, large scale discovery science.…”

mentioning

confidence: 99%

Enabling Coupled Quantitative Genomics and Proteomics Analyses from Rat Spinal Cord Samples

Butt

Pfeifer

Delaney

et al. 2007

Molecular & Cellular Proteomics

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Translational research is progressing toward combined genomics and proteomics analyses of small and precious samples. In our analyses of spinal cord material, we systematically evaluated disruption and extraction techniques to determine an optimum process for the coupled analysis of RNA and protein from a single 5-mm segment of tissue. Analyses of these distinct molecular species were performed using microarrays and high resolution two-dimensional gels, respectively. Comparison of standard homogenization with automated frozen disruption (AFD) identified negligible differences in the relative abundance of genes (44) with all genes identified by either process. Analysis on either the Affymetrix or Applied Biosystems Inc. gene array platforms provided good correlations between the extraction techniques. In contrast, the AFD technique enabled identification of more unique proteins from spinal cord tissue than did standard homogenization. Furthermore use of an optimized CHAPS/ urea extraction provided better protein recovery, as shown by quantitative two-dimensional gel analyses, than did solvent precipitation during TRIzol-based RNA extraction. Thus, AFD of tissue samples followed by protein and RNA isolation from separate aliquots of the frozen powdered sample is the most effective route to ensure full, quantitative analyses of both molecular entities.

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“…Proteins are excellent targets in disease diagnostics, prognostics, and therapeutics. Consequently, proteomic approaches (such as two-dimensional gel electrophoresis (2D-E), two-dimensional liquid chromatography (2-DL), and mass spectrometry (MS), which allow the simultaneous measurement and comparison of the expression levels of hundreds of proteins, represent powerful tools for (a) the discovery of novel hormone/drug targets and biomarkers and (b) studies of cellular metabolisms and protein expression (Righetti et al, 2004, Vlahou et al, 2005. Increasingly, proteomic techniques are being adopted to solve analytical problems and obtain a more comprehensive identification and characterization of molecular events associated with pathophysiological conditions.…”

Section: Proteomicsmentioning

confidence: 99%

New Challenges for Old Diseases: The Impact of -Omics Technologies in the Understanding of Allergic Diseases

Cárdaba¹,

Aguerri²,

Calzada³

et al. 2012

Allergic Diseases - Highlights in the Clinic, Mechanisms and Treatment

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Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches

Cited by 95 publications

References 34 publications

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

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

Enabling Coupled Quantitative Genomics and Proteomics Analyses from Rat Spinal Cord Samples

New Challenges for Old Diseases: The Impact of -Omics Technologies in the Understanding of Allergic Diseases

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