Peer Reviewed: Prefractionation Techniques in Proteome Analysis

Righetti, Pier Giorgio; Castagna, Annalisa; Herbert, Ben R.

doi:10.1021/ac012465t

Cited by 100 publications

(52 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Yet even these extreme measures might not suffice if one aims at full resolution of the proteome of a cell or tissue. The key to success would appear to be prefractionation, either chromatographic [44][45][46][47][48] or electrophoretic [6,49]. Reduction of the initial complexity found in an entire cell lysate, coupled with, e.g., narrow range focusing gels, appears to be the winning strategy since, on the one hand, it reduces the initial sample polydispersity, thus minimizing the chances of spot overlap and, in the other hand, it allows focusing of a narrow pI range sample (such as obtained in multicompartment electrolyzers) onto IPG ranges encompassing the same pI distribution.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, largescale analysis of proteomes should also take into account post-translational modifications (PTMs), since altered protein profiles of tissues or cells are the result of altered protein modification rather than altered gene expression [1,3,4]. To date, 2-D PAGE is the method of choice in proteomics for protein separation [1][2][3][5][6][7][8]; in addition, highly sophisticated technologies, i.e. MALDI peptide mass mapping and peptide sequencing by nanoelectrospray MS/MS and LC-MS/MS, have been developed to allow protein identification [1,3,5].…”

Section: General Aspectsmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2003

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: General Aspectsmentioning

confidence: 99%

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

et al. 2003

Self Cite

View full text Add to dashboard Cite

show abstract

“…Furthermore, these approaches often lead to fractions of such specificity that they can be separated with 1-D SDS-PAGE alone. Several commercial devices are now available for prefractionating proteins, including Rotofor (Bio-Rad, Hercules, CA, USA), IsoPrime (Amersham Biosciences, Uppsala, Sweden) and Gradiflow (Gradipore, Frenchs Forest, Australia), as well as several more that are described in the literature or in development [27][28][29]. Such devices will be essential for examining complex protein mixtures of not only simple unicellular organisms, but also samples from complex eukaryotic tissues and biological fluids.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2003

View full text Add to dashboard Cite

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.

show abstract

“…Aware of this new trend, the MCE with isoelectric membranes was miniaturized, so as to adapt it to proteome prefractionation with minute sample amounts [15]. The new instrument was shown to perform quite well in collecting proteome subfractions of very precise pI intervals, void of contamination from adjacent pI species [16][17][18]. An interesting variant of this approach is off-gel IEF in multicompartment devices [19][20][21].…”

Section: Introductionmentioning

confidence: 99%

Gel‐free IEF in a membrane‐sealed multicompartment cell for proteome prefractionation

et al. 2007

Self Cite

View full text Add to dashboard Cite

A minidevice for performing gel‐free proteome prefractionation via conventional IEF in soluble carrier ampholyte buffers is reported here. It consists of a compact block of polyoxymethylene in which eight samples and two electrode chambers are machined. Each of the eight sample chambers can be filled with up to 120 μL of sample and has the following size: 7 mm width, 3 mm depth and 10 mm height. The anodic and cathodic compartments have the same width and height as the sample chambers, but with a depth of 6 mm, thus accepting up to 250 μL of electrodic solutions. Focusing is in general accomplished in 2 h with a voltage gradient of up to 1000 V (7 cm electrode distance). Easy fractionation and collection of the content of the eight chambers is achieved by simply pressing a rubber diaphragm against the edges of the thin walls separating each well, this automatically breaking liquid continuity. The performance of this device has been tested by subfractionating total cell lysates of a human cancer cell line (U2Os) and of Escherichia coli bacterial cells, and by analysing the content of each chamber by mono‐dimensional SDS‐PAGE and 2‐D maps.

show abstract

Peer Reviewed: Prefractionation Techniques in Proteome Analysis

Cited by 100 publications

References 19 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

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

Gel‐free IEF in a membrane‐sealed multicompartment cell for proteome prefractionation

Contact Info

Product

Resources

About