Towards higher resolution: Two-dimensional Electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients

Wildgruber, Robert; Harder, Alois; Obermaier, Christian; Boguth, Günther; Weiss, Walter; Fey, Stephen J.; Larsen, Peter Mose; Görg, Angelika

doi:10.1002/1522-2683(20000701)21:13<2610::aid-elps2610>3.3.co;2-8

Cited by 44 publications

(64 citation statements)

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“…This is the topic of the present paper: a computer simulation procedure is described to quantitatively estimate the degree of overlapping present in the map. These informations are useful (i) for estimating the degree of error associated with identification and quantitation of each protein; (ii) for setting-up experimental conditions, i.e., zoom or narrow-pH range gels or enrichment/prefractionation strategies, which will increase resolution and greatly decrease the probability of spot overlapping [13][14][15][16][17].…”

Section: General Aspectsmentioning

confidence: 68%

“…In fact, if each band contains several proteins, the result is a list of the proteins present in the starting sample: quantitative data cannot be derived. Many efforts have been spent for achieving the highest resolution in protein separation, especially for crowded areas in 2-D gels: narrow immobilized pH gradients have been used, i.e., one strip 18 cm long covering 1 pH range [13][14][15] or the separation distance in the first dimension has been increased, i.e. from 8 cm to 11, 18, or even 39 cm [16,17].…”

Section: General Aspectsmentioning

confidence: 99%

“…In narrow-range IPG separations one pH unit is covered between acid and basic zones [13,14]: the resolution obtained in pH units is significantly higher (3 rd and 4 th column in Table 1). The data displayed in Table 1 highlight the wide range of pH resolution which can be experimentally obtained: from the worst case (i.e., 0.1 npH in a 7 cm strip of broad range gel with standard scanner) to the best one (i.e., 0.001 npH in a 39 cm strip of narrow range gel with a high resolution scanner).…”

Section: Spot Overlapping: Mathematical Computationmentioning

confidence: 99%

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Spot overlapping in two‐dimensional polyacrylamide gel electrophoresis maps: Relevance to proteomics

et al. 2003

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Proteomics requires a large-scale, simultaneous separation of proteins from a mixture, assessment of the relative abundance of these molecules, and identification and characterization of each component. In 2-D PAGE separations, the best method of choice for protein analysis, separation of all the proteins present in the sample is still far to be achieved and comigrating proteins in the same spot are in general present. A statistical estimation of the degree of spot overlapping present in a 2-D PAGE separation is here described: for different conditions of spot overcrowding in the map, the degree of overlapping can be quantified in terms of purity degree of each spot or percentage of proteins that will appear in the map as a single spot. A computer simulation approach is described: it is based on the protein separation pattern present in the experimental maps. The results thus obtained are compared to a theoretical model (statistical degree of peak overlapping model) based on random spot position. The described procedures were applied to an experimental reference map of human plasma. The severity of spot overlapping in 2-D PAGE maps is estimated and the influence of different experimental conditions (strip dimension, detector system performance, pI range) is discussed. These informations are useful to quantitatively estimate the degree of error associated with identification and quantitation of each protein and to set-up experimental conditions which will increase resolution and greatly decrease the probability of spot overlapping.

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Section: General Aspectsmentioning

confidence: 68%

Section: General Aspectsmentioning

confidence: 99%

Section: Spot Overlapping: Mathematical Computationmentioning

confidence: 99%

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Spot overlapping in two‐dimensional polyacrylamide gel electrophoresis maps: Relevance to proteomics

et al. 2003

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“…The use of multiple, narrow IEF gradient gels is one method for detecting more proteins. 44 Parallel examination of prefractionated cytosolic, membrane, and nuclear components would increase the Figure 7 Heat map of classification proteins. Proteins in the classification cluster with expression levels represented in red for low expression and green for higher expression.…”

Section: Discussionmentioning

confidence: 99%

Distinct proteomic profiles of amphetamine self-administration transitional states

et al. 2005

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In the rat, continuous access to d-amphetamine (d-AMPH) leads to lengthy bouts of self-administration, voluntary abstinence, and relapse to selfadministration. Previous studies have revealed that the progression from psychostimulant self-administration to abstinence to relapse is mediated in part by the ventral hippocampus. Stimulation of the ventral subiculum (vSub) during voluntary abstinence from d-AMPH self-administration reinstates self-administration and increases nucleus accumbens (NAc) dopamine efflux. Quantitative proteomic examination of the hippocampus from rats naïve to amphetamine, during a self-administration session 'Binge', during voluntarily abstinence 'Abstinent', and after reinstatement of selfadministration 'Relapse', revealed a differential proteomic state during abstinence. Actin-and cytoskeletal-related proteins were over-represented in the changes occurring during abstinence and suggest a decrease in actin filament polymerization. These changes may underlie alterations in neuronal tone during abstinence that could affect both neurotransmission and behavior. These data provide the first classification of addiction-related behaviors based on clustering of quantitative proteomic measurements.

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“…For example, highly hydrophobic membrane proteins are biologically designed to be insoluble in solution, therefore they remain nearly impossible to solubilize for electrophoretic purposes. Similarly, many low abundance proteins are present on 2-D gels, but they cannot be visualized due to the overwhelming presence of abundant 'housekeeping' proteins, and may only become visible when separated in conjunction with narrow range IPGs [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Strategies for the enrichment and identification of basic proteins in proteome projects

et al. 2003

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Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI . 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) -tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI . 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.

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Towards higher resolution: Two-dimensional Electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients

Cited by 44 publications

References 0 publications

Spot overlapping in two‐dimensional polyacrylamide gel electrophoresis maps: Relevance to proteomics

Spot overlapping in two‐dimensional polyacrylamide gel electrophoresis maps: Relevance to proteomics

Distinct proteomic profiles of amphetamine self-administration transitional states

Strategies for the enrichment and identification of basic proteins in proteome projects

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