Preparative two‐dimensional gel electrophoresis of membrane proteins

Pasquali, Christian; Fialka, Irene; Huber, Lukas A.

doi:10.1002/elps.1150181413

Cited by 136 publications

(126 citation statements)

References 34 publications

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“…Silver nitrate staining according to the protocol of Pasquai et al (16) and Coomassie Brilliant Blue R-250 (0.25% Brilliant Blue, 10% v/v acetic acid, 50% v/v methanol, destained in 20% v/v EtOH, 10% v/v acetic acid) was used for the analytical and micropreparative gels, respectively. The two-dimensional electrophoresisl images were captured with a ImageScanner (Amersham Pharmacia Biotech).…”

Section: Methodsmentioning

confidence: 99%

Proteomic Analysis of Human Acute Leukemia Cells

Cui

Wang

et al. 2004

Clinical Cancer Research

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Purpose: French-American-British (FAB) classification of acute leukemia with genetic heterogeneity is important for treatment and prognosis. However, the distinct protein profiles that contribute to the subtypes and facilitate molecular definition of acute leukemia classification are still unclear.Experimental Design: The proteins of leukemic cells from 61 cases of acute leukemia characterized by FAB classification were separated by two-dimensional electrophoresis, and the differentially expressed protein spots were identified by both matrix-assisted laser desorption/ioniza- tion-time-of-flight-mass spectrometry (MALDI-TOF-MS) and tandem electrospray ionization MS (ESI-MS/MS).Results: The distinct protein profiles of acute leukemia FAB types or subtypes were successfully explored, including acute myeloid leukemia (AML), its subtypes (M2, M3, and M5) and acute lymphoid leukemia (ALL), which were homogeneous within substantial samples of the respective subgroups but clearly differed from all other subgroups. We found a group of proteins that were highly expressed in M2 and M3, rather than other subtypes. Among them, myeloidrelated proteins 8 and 14 were first reported to mark AML differentiation and to differentiate AML from ALL. Heat shock 27 kDa protein 1 and other proteins that are highly expressed in ALL may play important roles in clinically distinguishing AML from ALL. Another set of proteins up-regulated was restricted to granulocytic lineage leukemia. High-level expression of NM23-H1 was found in all but the M3a subtype, with favorable prognosis.Conclusions: These data have implications in delineating the pathways of aberrant gene expression underlying the pathogenesis of acute leukemia and could facilitate molecular definition of FAB classification. The extension of the present analysis to currently less well-defined acute leukemias will identify additional subgroups.

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Section: Methodsmentioning

confidence: 99%

Proteomic Analysis of Human Acute Leukemia Cells

Cui

Wang

et al. 2004

Clinical Cancer Research

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“…Two-dimensional gel electrophoresis was performed with non-linear immobilized pH gradient strips, pH range 3-10 (Amersham Pharmacia Biotech, Uppsala, Sweden) as described (36,37).…”

Section: Methodsmentioning

confidence: 99%

Cross-talk between Caveolae and Glycosylphosphatidylinositol-rich Domains

Abrami¹,

Fivaz²,

Kobayashi³

et al. 2001

Journal of Biological Chemistry

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Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.

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“…We followed methods recently described by Rabilloud et al (26) and Pasquali et al (22), which show that the inclusion of thiourea in the extraction buffer significantly expands the range of integral membrane proteins that can be analyzed by 2D PAGE. Our preliminary work confirmed that thiourea dramatically increased the number of spots visible on 2D gels of PM preparations (data not shown).…”

Section: Analysis Of Pm Proteomesmentioning

confidence: 99%

Insights into a plasma membrane signature

Harvey

ZHANG

Landry

et al. 2001

Physiological Genomics

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. Insights into a plasma membrane signature. Physiol Genomics 5: [129][130][131][132][133][134][135][136] 2001.-The plasma membrane (PM) is an organized biological system that serves as a structural barrier and communication interface with the extracellular environment. Many basic questions regarding the PM as a system remain unanswered. In particular, we do not understand the scope of similarity and differences in protein expression at the PM. This study takes an initial step toward addressing these questions by comparing the PM proteomes of fibroblasts and mammary carcinoma cells. Three sets of proteins were revealed by the study. The first set comprises between 9 and 23% of all proteins at the PM and appears to be common to both fibroblasts and mammary carcinoma. A second group of proteins, comprising ϳ40% of the proteins at the PM, is tightly linked to cell lineage. The third set of proteins is unique to each cell line and is independent of cell lineage. It is reasonable to hypothesize then, that this third group of proteins is responsible for unique aspects of cell behavior. In an effort to find proteins linked to the metastatic phenotype, we identified several proteins that are uniquely expressed at the PM of the metastatic MDA-MB-435 cells. These proteins have functions ranging from cell adhesion to the regulation of translation and the control of oxidant stress.proteome; expression profiling; breast cancer; metastasis AS EARLY as 1953, Nordling (19) suggested that the clinical manifestations of cancer result from the cumulative effect of multiple genetic mutations. The multifactorial nature of tumor progression precludes its explanation by any single factor or even a small group of factors. It has yet to be determined how many alterations must occur to convert a normal mammary epithelial cell to carcinoma or how many additional changes are necessary for the carcinoma to progress to invasive disease. Answers to these questions require global examinations of biological systems.This study focuses on the plasma membrane (PM) as a system. The PM regulates many aspects of tumor progression including cellular differentiation, cell proliferation, adhesion and migration, pericellular proteolysis, and even the escape from immune surveillance. Each of these events represents a point for potential therapeutic intervention, making the PM a rich source of drug targets. Many of the molecules at the PM are also exposed to the extracellular milieu, providing an additional therapeutic advantage. As recognized more than 25 years ago by Garth Nicolson, there is a great need to define the PM as a system: "It would be convenient if these (biological) properties were definable by unique cell surface characteristics which were readily identifiable so that highly specific molecular approaches to fighting cancer could be developed" (18).Presented with this long-recognized but unmet need, the present study was aimed at gaining some basic information on the PM proteome of mammary carcinoma. We compared the PM proteomes of mammary...

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Preparative two‐dimensional gel electrophoresis of membrane proteins

Cited by 136 publications

References 34 publications

Proteomic Analysis of Human Acute Leukemia Cells

Proteomic Analysis of Human Acute Leukemia Cells

Cross-talk between Caveolae and Glycosylphosphatidylinositol-rich Domains

Insights into a plasma membrane signature

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