Analysis of the Biocompatibility of Different Intraocular Lens (IOL) Material Using Mass Spectrometry Tisssue Imaging

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 ▲]

Surface and bio-adhesion properties of new hydrophobic and current materials for artificial intraocular lens

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 ▲]

An analytical pipeline for MALDI in-source decay mass spectrometry imaging

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 ▲]

2D 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.

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 ▲]

MALDI MS Tissue Imaging of Crystallins using an original metyhod to direct protein identification on lens slices

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 ▲]

MCF-7/BOS cells membrane proteome: comparison of two isolation methods using mass spectrometry

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 ▲]

Global strategy for the development of estrogenic compounds detection screening test

Poster (2007, May)