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See detailRGD surface functionalization of the hydrophilic acrylic intraocular lens material to control posterior capsular opacification
Huang, Yi-Shiang ULg; Bertrand, Virginie ULg; Bozukova, Dimitriya et al

in PLoS ONE (in press)

Posterior Capsular Opacification (PCO) is the capsule fibrosis developed on implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LECs) undergoing Epithelial Mesenchymal ... [more ▼]

Posterior Capsular Opacification (PCO) is the capsule fibrosis developed on implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LECs) undergoing Epithelial Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including the patient’s age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs have shown that the former has more severe PCO. On the other hand, we have previously demonstrated that the adhesion of LECs is favored on hydrophobic compared to hydrophilic materials. By combining these two facts and contemporary knowledge in PCO development via the EMT pathway, we propose a biomimetically inspired strategy to promote LEC adhesion without de-differentiation to reduce the risk of PCO development. By surface grafting of a cell adhesion molecule (RGD peptide) onto the conventional hydrophilic acrylic IOL material, the surface-functionalized IOL can be used to reconstitute a capsule-LEC-IOL sandwich structure, which has been considered to prevent PCO formation in literature. Our results show that the innovative biomaterial improves LEC adhesion, while also exhibiting similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties compared to the starting material. In addition, compared to the hydrophobic IOL material, our bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression, which is the crucial pathway to induce PCO. The in vitro assays suggest that this biomaterial has the potential to reduce the risk factor of PCO development. [less ▲]

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See detailBiointerface multiparametric investigation of intraocular lens acrylic materials
Bertrand, Virginie ULg; Bozukova, Dimitriya; Svaldo Lanero, Tiziana et al

in Journal of Cataract & Refractive Surgery (2014), 40(9), 1536-1544

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See detailComparison of two FFPE preparation methods using label-free shotgun proteomics: Application to tissues of diverticulitis patients.
Quesada-Calvo, Florence; Bertrand, Virginie ULg; Longuespée, Rémi ULg et al

in Journal of proteomics (2014), 112C

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic ... [more ▼]

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic biomarkers. However, the critical step in this field is the ability to obtain an efficient and repeatable extraction using the limited quantities of material available for research in hospital biobanks. This work describes the evaluation of the peptide/protein extraction using FFPE sections treated by the following two methods before shotgun proteomic analysis: a commercial solution (FFPE-FASP) (filter aided sample preparation) and an antigen retrieval-derived protocol (On Slice AR). Their efficiencies and repeatabilities are compared using data-independent differential quantitative label-free analysis. FFPE-FASP was shown to be globally better both qualitatively and quantitatively than On Slice AR. FFPE-FASP was tested on several samples, and differential analysis was used to compare the tissues of diverticulitis patients (healthy and inflammatory tissues). In this differential proteomic analysis using retrospective clinical FFPE material, FFPE-FASP was reproducible and provided a high number of confident protein identifications, highlighting potential protein biomarkers. BIOLOGICAL SIGNIFICANCE: In clinical proteomics, FFPE is an important resource for retrospective analysis and for the discovery of biomarkers. The challenge for FFPE shotgun proteomic analysis is preparation by an efficient and reproducible protocol, which includes protein extraction and digestion. In this study, we analyzed two different methods and evaluated their repeatabilities and efficiencies. We illustrated the reproducibility of the most efficient method, FFPE-FASP, by a pilot study on diverticulitis tissue and on FFPE samples amount accessible in hospital biobanks. These data showed that FFPE is suitable for use in clinical proteomics, especially when the FFPE-FASP method is combined with label-free shotgun proteomics as described in the workflow presented in this work. [less ▲]

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See detailBiointerface multiparametric study of intraocular lens acrylic materials.
Bertrand, Virginie ULg; Bozukova, Dimitriya; Svaldo Lanero, Tiziana et al

in Journal of cataract and refractive surgery (2014), 40(9), 1536-44

PURPOSE: To compare hydrophilic and hydrophobic acrylic materials designed for intraocular lenses in a multiparametric investigation in a liquid environment to highlight their properties in terms of ... [more ▼]

PURPOSE: To compare hydrophilic and hydrophobic acrylic materials designed for intraocular lenses in a multiparametric investigation in a liquid environment to highlight their properties in terms of adhesion forces, lens epithelial cell (LEC) adhesion, and tissue response as indicators of the risk for posterior capsule opacification (PCO) development. SETTING: University of Liege, Liege, Belgium. DESIGN: Experimental study. METHODS: The hydrophobicity and surface adhesion force were assessed using contact-angle and atomic force microscopy measurements. The bioadhesiveness of the disks and the tissue response were determined by in vitro experiments using bovine serum albumin and porcine LECs and by in vivo rabbit subcutaneous implantation, respectively. RESULTS: Increasing surface hydrophobicity led to a greater surface-adhesion force and greater LEC adhesion. After 1 month, the rabbit subcutaneous implants showed a similar thin layer of fibrous capsule surrounding the disks without extensive inflammation. A layer of rounded cells in contact with disks was detected on the hydrophobic samples only. CONCLUSIONS: Hydrophobic acrylic disks that have been associated with a reduced risk for PCO in clinical studies showed increased tackiness. FINANCIAL DISCLOSURES: Proprietary or commercial disclosures are listed after the references. [less ▲]

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See detailBioactive Intraocular Lens – A Strategy to Control Secondary Cataract
Huang, Yi-Shiang ULg; Bertrand, Virginie ULg; Bozukova, Dimitriya et al

in IFMBE Proceedings (2014), 41

Cataract is the opacity of the lens, causing impairment of vision or even blindness. Today, a surgery is still the only available treatment. The intraocular lens (IOL) is a polymer implant designed to ... [more ▼]

Cataract is the opacity of the lens, causing impairment of vision or even blindness. Today, a surgery is still the only available treatment. The intraocular lens (IOL) is a polymer implant designed to replace the natural lens in the cataract surgery. However, the bioinert materials could not satisfy the unmet need in the secondary cataract control. Posterior capsular opacification (PCO, or Secondary Cataract), characterized by a thick and cloudy layer of lens epithelial cells (LECs), is the most common postoperative complication. In our research, a bioactive molecule is immobilized onto the conventional acrylic hydrophilic polymer pHEMA (Poly(2-hydroxyethyl methacrylate)) using oxygen plasma treatment followed by deposition. The RGD peptide sequence, being well-known for its ability to promote cellular attachment by binding to integrin receptors, is designed to stimulate the adhesion of LECs on the IOL. Our data show the peptide immobilized biomaterial not only exhibits similar optical property, but also reveals enhanced biological properties in cell adhesion and cell morphology maintenance. By means of surface functionalization of IOL to stimulate LECs adhesion, the secondary cataract could be controlled. [less ▲]

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See detailPolymer based intraocular lens adsorbome: a bottom up proteomics study
Bertrand, Virginie ULg; Huang, Yi-Shiang ULg; Mazzucchelli, Gabriel ULg et al

Conference (2013, September 08)

In the present work an optimized sample preparation protocol to identify and quantify the “adsorbomes” of hydrophilic and hydrophobic materials for IOLs known to have a higher or a lower incidence of PCO ... [more ▼]

In the present work an optimized sample preparation protocol to identify and quantify the “adsorbomes” of hydrophilic and hydrophobic materials for IOLs known to have a higher or a lower incidence of PCO, respectively was obtained. [less ▲]

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See detailIntraocular Lens Adsorbome: a Proteomic Study of Adsorbed Proteins onto Acrylic Materials and Its Implication in Secondary Cataract
Huang, Yi-Shiang ULg; Bertrand, Virginie ULg; Mazzucchelli, Gabriel ULg et al

Poster (2012, September 17)

The intraocular lens (IOL) is a polymer implant designed to replace the natural lens after cataract surgery. When the implant is introduced into the lens capsule, the polymer starts to interact with the ... [more ▼]

The intraocular lens (IOL) is a polymer implant designed to replace the natural lens after cataract surgery. When the implant is introduced into the lens capsule, the polymer starts to interact with the aqueous humour and the exchange of molecules between the solid and the liquid begins. The nature of exchange in water, ions, and biomolecules may result in several postoperative complications including glistening, calcification, and posterior capsular opacification. The posterior capsular opacification (PCO, also called “Secondary Cataract”) is raised from the over-growth of residual lens epithelial cells. The first step of the over-growth process of the cells is their adhesion to the deposited biomolecules, such as proteins involved in extra-cellular matrices. The purpose of this study is to identify the principal proteins adsorbed onto the acrylic polymers by mass spectrometry. The concept of adsorbome is to generate a list of adsorbed proteins to the hydrophilic and hydrophobic polymers, and then compare the difference to the original component of aqueous humour in order to see the affinity of individual protein to each material. Two kinds of hydrophilic and two kinds of hydrophobic acrylic polymers were tested for their adsorbomes by treating them with an aqueous humour analogue and the major adsorbed proteins were identified by mass spectrometry. Interestingly, the hydrophilic acrylic polymer shows a relative lower protein adsorption rate but shows a higher incidence of secondary cataract. This phenomenon implies the adsorbed proteins play a crucial role in progress of secondary cataract. [less ▲]

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See detailAnalysis of the Biocompatibility of Different Intraocular Lens (IOL) Material Using Mass Spectrometry Tisssue Imaging
Bertrand, Virginie ULg; Debois, Delphine ULg; Calligaris, David ULg et al

Conference (2012, September 04)

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract ... [more ▼]

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract. The surgical intervention consists in removing the cloudy lens and to replace it by an artificial intraocular lens (IOL). The in vivo implantation of these synthetic lenses involves the evaluation of several factors as their physico-chemical properties, their capacities to interact with lens epithelial cells and proteins, as well as their biocompatibility. During a previous study, we demonstrated major differences concerning the tackiness (atomic force microscopy), the cellular adhesion and the protein adsorption of various polymer disks intended for the manufacturing of intraocular lenses. The aim of this work was to correlate a histological analysis to a mass spectrometry imaging analysis performed on the same sample. To estimate the biocompatibility of the biomaterials, an animal testing was realized in rabbits. The various polymers were implanted subcutaneously. After one month, the 2 cm x 3 cm pieces of rabbit skin and underlying muscle with a 2 cm thickness were removed, fixed with formaldehyde 10% during six days, treated for the paraffin inclusion and stored at room temperature until use. Slices of 5 µm thickness were performed using a microtome. Paraffin was removed and tissue sections were washed in graded ethanol baths. The slices were then stained with the hematoxylin and eosin dyes. The analysis of stained sections showed different histo-morphological features according to the implanted polymer. For MALDI MSI purposes, on tissue protein digestion was performed using trypsin (1) and the MALDI matrix (α-cyanohydroxycinnamic acid, 5 mg/mL in ACN/0.2% TFA 70:30) was deposited using an ImagePrep automated sprayer (Bruker Daltonics, Bremen, Germany). Experiments were carried out using an UltraFlex II TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). MALDI imaging can show the detection of different proteomic profiles according to the tested biomaterials, which may be considered as biocompatibility markers. The MALDI images of these markers are then correlated with the histo-morphological profiles. Consequently, mass spectrometry imaging can become a powerful tool in the evaluation of the biocompatibility of artificial implants in biomedical application. [less ▲]

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See detailIdentification and quantification of concentration-dependent biomarkers in MCF-7/BOS cells exposed to 17β-estradiol by 2-D DIGE and label-free proteomics
Collodoro, Mike ULg; Lemaire, Pascale ULg; Eppe, Gauthier ULg et al

in Journal of Proteomics (2012), in press

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E2). The biomarkers were identified using 2 independent and complementary techniques, 2 ... [more ▼]

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E2). The biomarkers were identified using 2 independent and complementary techniques, 2-D DIGE / MALDI-TOF peptide mass fingerprint, and 2-D UPLC-ESI MS/MS. These markers form a preliminary molecular signature that can be used when testing the estrogenic activity of xenobiotics, either pure or in mixtures. [less ▲]

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See detailSurface and bio-adhesion properties of new hydrophobic and current materials for artificial intraocular lens
Bertrand, Virginie ULg; Svaldo Lanero, Tiziana ULg; Duwez, Anne-Sophie ULg et al

Poster (2012)

A high bio-adhesion appears to be one of the key factor for posterior capsular opacification (PCO) prevention. Indeed, the proteins adsorption and the lens epithelial cells (LEC) adhesion both contribute ... [more ▼]

A high bio-adhesion appears to be one of the key factor for posterior capsular opacification (PCO) prevention. Indeed, the proteins adsorption and the lens epithelial cells (LEC) adhesion both contribute to PCO development. We present in this work the comparison of a new glistening free hydrophobic material (GF® from Physiol) with benchmark hydrophobic and hydrophilic materials regarding their chemicophysical properties and their respective ability to interact with lens epithelial cells and proteins. For this purpose, we determined the hydrophobicity by contact angle measurement (assessed by water drop and air bubble methods), the surface adhesiveness by atomic force microscopy (AFM), the proteins adsorption by fluorescent measurement and the LEC adhesion by the determination of cell density. The new hydrophobic material presents comparable hydrophobicity, proteins adsorption and LEC adhesion to current commercial hydrophobic material. Its adhesiveness, measured with the AFM, is intermediate between hydrophilic and hydrophobic materials. In conclusion, the bio-adhesion properties of this new glistening free hydrophobic IOL material are similar to generic hydrophobic acrylic materials and therefore should to the same extent prevent PCO. [less ▲]

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See detailAssessment of new-generation glistening-free hydrophobic acrylic intraocular lens material
Pagnoulle, Christophe; Bozukova, Dimitriya; Gobin, Laure et al

in Journal of Cataract & Refractive Surgery (2012), 38

To determine the hydrophobic, antiglistening, and bioadhesiveness properties of a new polymer, GF rawmaterial, and to determine the suitability of thismaterial for use in intraocular lenses (IOLs).

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See detailAn analytical pipeline for MALDI in-source decay mass spectrometry imaging
Zimmerman, Tyler ULg; Debois, Delphine ULg; Mazzucchelli, Gabriel ULg et al

in Analytical Chemistry (2011), 83(15), 6090-6097

In-source decay (ISD) fragmentation as combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allows protein sequencing directly from mass spectra. Acquisition of MALDI-ISD ... [more ▼]

In-source decay (ISD) fragmentation as combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allows protein sequencing directly from mass spectra. Acquisition of MALDI-ISD mass spectra from tissue samples is achieved using an appropriate MALDI matrix, such as 1,5-diaminonaphthalene (DAN). Recent efforts have focused on combining MALDI-ISD with mass spectrometry imaging (MSI) to provide simultaneous sequencing and localization of proteins over a thin tissue surface. Successfully coupling these approaches requires the development of new data analysis tools, but first, investigating the properties of MALDI-ISD as applied to mixtures of protein standards reveals a high sensitivity to the relative protein ionization efficiency. This finding translates to the protein mixtures found in tissues and is used to inform the development of an analytical pipeline for data analysis in MALDI-ISD MS imaging, including software to identify the most pertinent spectra, to sequence protein mixtures, and to generate ion images for comparison with tissue morphology. The ability to simultaneously identify and localize proteins is demonstrated by using the analytical pipeline on three tissue sections from porcine eye lens, resulting in localizations for crystallins and cytochrome c. The variety of protein identifications provided by MALDI-ISD-MSI between tissue sections creates a discovery tool, and the analytical pipeline makes this process more efficient. [less ▲]

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See detail2D DIGE, label free quantification, principal component and mass spectrometry analysis for biomarkers discovery in MCF-7/BOS cells exposed to 17β-estradiol and endocrine disruptors.
Collodoro, Mike ULg; Lemaire, Pascale; Eppe, Gauthier ULg et al

in Organohalogen Compounds (2011)

Endocrine system disruption has become a subject of great interest over the last few decades, since it has become evident that natural and also synthetic substances can mimic or reduce the activity of ... [more ▼]

Endocrine system disruption has become a subject of great interest over the last few decades, since it has become evident that natural and also synthetic substances can mimic or reduce the activity of endogenous hormones. Compounds with estrogenic activity are an important family of potential endocrine disruptors that have to be monitored either in the food chain or in the environment. Estrogens are known to induce or promote hormonal dependent cancers, to reduce sperm counts and fertility in men and generate the feminization of exposed wildlife populations. The rapid screening of unwanted chemicals in the food chain is beset by difficulties. The number of toxic compounds is very large and no universal method can cope with their diversity. In this work, emergent differential proteomic techniques are used to discover a set of biomarkers for the development of a multiple estrogen contaminants screening test. [less ▲]

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See detailMALDI-In Source Decay Applied to Mass Spectrometry Imaging: A New Tool for Protein Identification.
Debois, Delphine ULg; Bertrand, Virginie ULg; Quinton, Loïc ULg et al

in Analytical Chemistry (2010), 82(10), 3969-4304

Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is a powerful technique giving access to the distribution of a large range of biomolecules directly from a tissue section ... [more ▼]

Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is a powerful technique giving access to the distribution of a large range of biomolecules directly from a tissue section, allowing, for example, the discovery of new pathological biomarkers. Nevertheless, one main difficulty lies in the identification of the detected species, especially proteins. MALDI-in source decay (ISD) is used to fragment ions directly in the mass spectrometer ion source. This technique does not require any special sample treatment but only the use of a specific MALDI matrix such as 2,5-dihydroxybenzoic acid or 1,5-diaminonaphthalene. MALDI-ISD is generally employed on classical, purified samples, but here we demonstrate that ISD can also be performed directly on mixtures and on a tissue slice leading to fragment ions, allowing the identification of major proteins without any further treatment. On a porcine eye lens slice, de novo sequencing was even performed. Crystallins not yet referenced in databases were identified by sequence homology with other mammalian species. On a mouse brain slice, we demonstrate that results obtained with ISD are comparable and even better than those obtained with a classical in situ digestion. [less ▲]

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See detailMALDI MS Tissue Imaging of Crystallins using an original metyhod to direct protein identification on lens slices
Bertrand, Virginie ULg; Debois, Delphine ULg; Quinton, Loïc ULg et al

Poster (2010, April 16)

The lens is a transparent, biconvex structure in the eye that, along with the cornea, helps to refract light to be focused on the retina. Crystallins, α, β and γ, are the predominant structural proteins ... [more ▼]

The lens is a transparent, biconvex structure in the eye that, along with the cornea, helps to refract light to be focused on the retina. Crystallins, α, β and γ, are the predominant structural proteins in lens. They constitute 90% of water soluble proteins and contribute to its transparency and refractive properties by a uniform concentration gradient in the lens. Nevertheless, if these crystallins undergo post translational modifications, they become less soluble and the opacity of eye lens increases. This phenomenon defines cataract. Yet, the nature and the mechanism of occurring of these modifications and how they happen are not fully understood. MALDI mass spectrometry imaging is a recent technique allowing examining proteins in their native location without the need for traditional processing methods such as extraction, homogenization, and separation. Nevertheless, one main difficulty lies in the identification of the detected species, especially proteins. MALDI-In Source Decay (MALDI-ISD) is a fragmentation process occurring in the mass spectrometer ion source. When the analyzed sample is a protein, ISD fragmentation leads to b-, c- and z-ions series, which allows for some sequencing of the protein. One great advantage of ISD is its fastness and easiness to be implemented since there is no need for a special treatment of the sample. The only requirement is the use of “ISD-favourable MALDI matrix” such as 2,5-dihydroxybenzoic acid or 1,5-diaminonaphtalene. 18 µm-thick equatorial sections of frozen porcine eye lenses were realized with a cryostat. 1,5-DAN matrix was either manually deposited or sprayed with an ImagePrep automated device (Bruker Daltonics). Data were acquired with an UltraFlex II MALDI-TOF/TOF mass spectrometer (BD) in positive reflector mode. For imaging experiments, the surface of the sample was divided into 100-µm-wide pixels and 500 shots were averaged on each. Based on calculated mass differences between consecutive ISD fragments peaks, tags of amino acids were established and submitted to a search in protein databases using a BLAST algorithm (search by sequence homology). Imaging experiments showed that the localization information may be very useful to associate fragments which exhibit close distributions, suggesting they are originating from the same protein. It is thus possible to arrange fragments in groups of probable origin and to extract the mass spectrum of a high-intensity pixel. This allows to work with a “purified” ISD mass spectrum where fragments of only one protein are present and potentially exhibiting a higher number of peaks, leading to a longer tag and to an easier identification. With this imaging strategy, we were able to identify (by homology) the Beta-Crystallins S and B2, the Gamma-Crystallin B, the Alpha-Crystallin A. [less ▲]

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See detailIdentification of biomarkers to estrogen exposure using MCF-7/BOS cell line exposed to 17β-estradiol and phytoestrogens
Collodoro, Mike ULg; Bertrand, Virginie ULg; Lemaire, Pascale ULg et al

Poster (2009, June)

Use of an estrogen responsive cell line and proteomic for biomarker discovery and the screening of xenoestrogen

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See detailMCF-7/BOS cells membrane proteome: comparison of two isolation methods using mass spectrometry
Bertrand, Virginie ULg; Massart, Anne-Cécile ULg; De Pauw, Marie-Claire ULg et al

Poster (2009)

Membrane proteins play an important role in biological processes but their isolation and quantification using classical techniques is often limited due to their poor solubility and relatively low ... [more ▼]

Membrane proteins play an important role in biological processes but their isolation and quantification using classical techniques is often limited due to their poor solubility and relatively low abundance. These membrane markers have to be accessible to antibodies and should be potential therapeutic targets. The tests were conducted on MCF-7 / BOS cell line, immortal and easier to cultivate. The goal of this work is to obtain a pure membrane fraction to facilitate the analysis of the sample. To isolate transmembrane proteins, we compared two methods. The first one used different extraction cycles characterized by different buffers to isolate membrane proteins. The second method labelled accessible extracellular domains at the surface of MCF-7 cells with biotin prior to differential centrifugation. [less ▲]

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See detailMCF-7/BOS cells membrane proteome: comparison of two isolation methods using mass spectrometry
Bertrand, Virginie ULg; Massart, Anne-Cécile ULg; De Pauw, Marie-Claire ULg et al

Poster (2009)

Membrane proteins (MP) play an important role in biological processes. Isolation and quantification of these MP using classical techniques is often limited due to their poor solubility and relatively low ... [more ▼]

Membrane proteins (MP) play an important role in biological processes. Isolation and quantification of these MP using classical techniques is often limited due to their poor solubility and relatively low abundance. These membrane proteins enclosed markers which could be potential therapeutic targets. These potential therapeutic targets have to be accessible to antibodies and need to be presented in the plasmic membrane. Assays were conducted on MCF-7 / BOS cell line, immortal and easier to cultivate. The goal of this work is to obtain an enriched membrane fraction to facilitate the analysis of the sample and to simplify the complex proteins mixture. To isolate transmembrane proteins, we compared two methods. The first one used different extraction cycles characterized by different buffers to isolate membrane proteins. The second method labelled accessible extracellular domains at the surface of MCF-7 cells with biotin prior to differential centrifugation. The obtained enriched membrane proteome was digested with trypsin and/or Lysyl Endopeptidase. Obtained peptides were separated by 2D-HPLC chromatography and on-line analysed ion trap mass spectrometer, the Esquire HCT. [less ▲]

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