Two-dimensional maps in soft immobilized pH gradient gels: A new approach to the proteome of the Third Millennium

Candiano, Giovanni; Musante, Luca; Bruschi, Maurizio; Ghiggeri, Gian Marco; Herbert, Ben R.; Antonucci, Francesca; Righetti, Pier Giorgio

doi:10.1002/1522-2683(200202)23:2<292::aid-elps292>3.0.co;2-5

Cited by 47 publications

(31 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Existing 2-DE technology is limited in its ability to separate high molecular mass proteins due to gel pore sizes in both the first and second dimension acrylamide matri-ces [12][13]. Likewise, basic proteins remain a technical challenge to separate, while those at extremely alkaline pH (pI .…”

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

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

show abstract

“…On the other hand, the difficulty to ensure large proteins transfer from the first to the second dimension is well known. In this way, Candiano et al nicely described the use of soft immobiline gels instead of commercial IPG strips to favor detection of large filamentous proteins [10,19]. In the present work, we show an alternative by which high molecular weight proteins, i.e.…”

Section: Combined Strategy: Hypotonic Lysis Isolation and Further Purmentioning

confidence: 57%

A novel methodology for the analysis of membrane and cytosolic sub‐proteomes of erythrocytes by 2‐DE

et al. 2009

View full text Add to dashboard Cite

A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DEWith the aim of studying a wide cohort of erythrocyte samples in a clinical setting, we propose here a novel approach that allows the analysis of both human cytosolic and membrane sub-proteomes. Despite their simple structure, the high content of hemoglobin present in the red blood cells (RBCs) makes their proteome analysis enormously difficult. We investigate here different strategies for isolation of the membrane and cytosolic fractions from erythrocytes and their influence on proteome profiling by 2-DE, paying particular attention to hemoglobin removal. A simple, quick and satisfactory approach for hemoglobin depletion based on HemogloBind TM reagent was satisfactorily applied to erythrocyte cells, allowing the analysis of the cytosolic sub-proteome by 2-DE without major interference. For membrane proteome, a novel combined strategy based on hypotonic lysis isolation and further purification on minicolumns is described here, allowing detection of high molecular weight proteins (i.e. spectrin, ankyrin) and wellresolved 2-DE patterns. An aliquot of the membrane fraction was also in solution digested and analyzed by nano-LC coupled to an LTQ-Orbitrap mass spectrometer. A total of 188 unique proteins were identified by this approach. This study sets the basis for future clinical studies where the erythrocyte cell may be implicated.

show abstract

“…The majority of proteins (∼40%) visualized possessed molecular weights between 25 and 37 kDa. Current commercially available 4% total acrylamide IPG gel strips limit the high molecular weight proteins visualized with 2-D electrophoresis (Candiano et al, 2002). As a natural consequence of the effect of a stress factor on a tissue, expression of genes of proteins protecting cells from damage is induced.…”

Section: Discussionmentioning

confidence: 99%

Tetracycline Accumulation in Pea Seedlings and its Effects on Proteome and Enzyme Activities

Margas¹,

Piotrowicz-Cieślak

Ziółkowska³

et al. 2016

IJAB

View full text Add to dashboard Cite

To cite this paper: Margas, M., A.I. Piotrowicz-Cieślak, A. Ziółkowska and B. Adomas, 2016. Tetracycline accumulation in pea seedlings and its effects on proteome and enzyme activities. AbstractAmong antibiotics, tetracyclines are the most commonly used and detected in the environment. In this study, the amount of tetracycline taken up from soil by pea seedlings was analyzed, identified its main site of accumulation in plants and determined also changes in the protein profile of pea. The study demonstrates that pea seedlings take up tetracycline from soil and transport the drug via roots to over-ground parts and then accumulate it in the youngest parts, such as upper stem and leaves. After the taken up of drug, the activity of guaiacol peroxidase is modified and changes in the profile of proteins, as determined by two-dimensional gel electrophoresis occur. The majority of proteins (∼40%) visualized possessed molecular weight between 25 and 37 kDa. Only 8% of the proteins had molecular weight lower than 20 kDa, and 2% greater than 75 kDa. The number of spots in the control samples was 194, which is less by 49 than at the concentration of 150 mg kg -1 of soil. Isoflavone reductase was present only in seedlings growing with tetracycline. Tetracycline uptake from soil modify mainly the changes in biochemical processes connected with protein.

show abstract

Two-dimensional maps in soft immobilized pH gradient gels: A new approach to the proteome of the Third Millennium

Cited by 47 publications

References 20 publications

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

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

A novel methodology for the analysis of membrane and cytosolic sub‐proteomes of erythrocytes by 2‐DE

Tetracycline Accumulation in Pea Seedlings and its Effects on Proteome and Enzyme Activities

Contact Info

Product

Resources

About