Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/20—Dermatological disorders
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/20—Dermatological disorders
  • G01N2800/205—Scaling palpular diseases, e.g. psoriasis, pytiriasis

Definitions

• the invention relates to the use of mass spectrometry from a non-invasive skin sample for the diagnosis, prognosis or therapeutic follow-up of cutaneous pathologies, such as psoriasis.
• the invention more particularly relates to a method for analyzing a sample of the upper layers of the epidermis. Diagnoses of cutaneous pathologies are usually performed by biopsy or by local examination of skin symptoms, such an examination involving the visual evaluation of areas showing inflammation.
• a non-invasive method, such as visual examination is more advantageous than biopsies in terms of cost, time and comfort for the patient.
• diagnostic errors can occur especially when the pathologies in question have only preliminary or poorly developed symptoms.
• the patient may have to deal with a succession of ineffective treatments, which is of course to be avoided.
• a succession of ineffective treatments may also result in the abandonment of any treatment by the patient.
• proteins are not necessarily related to the presence of messenger RNA encoding these proteins but may result from post-translational modifications (glycosylation or deglycosylation, glycation, oxidation, phosphorylation. ..) or be related to the fate of these proteins such as, for example, enzymatic hydrolysis.
• This hydrolysis by the action of endogenous proteases can lead to the appearance of mature forms or the disappearance of certain markers. These events are particularly important in the epidermal layers. These biochemical changes may sign an epidermal state (disorder) that can only be observed by the detection and identification of proteins including proteomic analysis.
• Patent application WO2007 / 96846 describes a skin evaluation method by measuring metabolites of skin samples. This method makes it possible to follow the metabolites of the cutaneous surface, which are the consequences and not the actors of the biochemical reactions that have occurred in the skin.
• the techniques developed in this application are specific to the analysis of the metabolome and are not suitable for proteome analysis.
• the person skilled in the art can not extrapolate this method of evaluation of the metabolome to the identification of protein markers.
• the identification of the protein content and its proteomic analysis makes it possible to follow specific markers of a cutaneous pathology and to guide the diagnosis.
• most of the therapeutic targets are of a protein nature, which reinforces the interest of discovering, identifying and tracking the proteins on the surface of the skin through a method specific to these.
• the identification of proteins upstream of the metabolome is therefore fundamental for the characterization of the tissue response.
• the present invention proposes to solve the disadvantages of the state of the art by presenting a new reliable, rapid and non-invasive method of collecting biological data useful for establishing a diagnosis or prognosis of a disease. skin pathology such as psoriasis.
• One aspect of the invention therefore relates to a non-invasive method of collecting biological data useful for establishing a diagnosis or prognosis of a particular skin pathology in a patient, characterized in that a) is taken a skin sample containing biological material, by applying a layer of adhesive to the skin of said patient, then removing said adhesive, b) detecting by mass spectrometry in the cutaneous sample obtained in a) at least one protein whose presence, absence, or variation in quantity or concentration with respect to a standard value is associated with the presence, evolution, or absence of a particular skin pathology.
• Mass spectrometry is already used in the identification of complex mixtures of chemical compounds and molecules of biological origin. Unlike tools conventionally used as chips or the ELISA technique, this tool makes it possible to visualize new markers, new proteins not yet identified and this by a method having a better sensitivity.
• the basic principle of mass spectrometry analysis is the measurement of the mass / charge ratio of ionic species that have been created by ionization of the sample to be analyzed and to which electric and magnetic fields are applied.
• One of the most frequently used ionization methods for biological samples is electro-fogging, known as "ElectroSpray” (ES), which generates multicharged ions and allows the use of inexpensive conventional analyzers such as quadrupoles.
• ES Electro-fogging
• the ionization source for the electrospray can be multiple, and in particular can provide good sensitivity for low flow.
• Another commonly used ionization method is matrix-assisted laser desorption ionization, known as
• MALDI-TOF is an ionization method well suited to flight time analyzers (MALDI for "Matrix Assisted Laser Desorption and Ionization” and TOF for "Time ofFHght").
• the mass / electric charge ratio is measured in different ways, for example by measuring the standard deviation of ionized species (“Sector MS") or the measurement of flight time (“TOF MS").
• Sample MS standard deviation of ionized species
• TOF MS flight time
• Today the methods of analysis are varied and include, for example, quadrupole or quadrupole mass analyzers (QMS), ion trap quadrupole mass analyzers (QIT), Fourier transform infrared spectrometry (FTICR) analyzers.
• QMS quadrupole or quadrupole mass analyzers
• QIT ion trap quadrupole mass analyzers
• FTICR Fourier transform infrared spectrometry
• tandem mass spectrometry is the combination of several analyzers that allows the selection of a particular ion then the identification of its fragments by successively inserting mass separation steps with fragmentation steps .
• a tandem analyzer can isolate a peptide resulting from trypsin hydrolysis, protein fragments or a protein from a complex mixture, and then stabilize it to allow its fragmentation (for example by means of a gas or another known method) and catalog the fragments thus produced.
• the use of data banks then makes it possible to identify the proteins present in the sample.
• the method according to the invention uses the tandem mass spectrometry (MS / MS) technique.
• a first fractionation method makes it possible to separate proteins from each other by two-dimensional electrophoresis in polyacrylamide gel.
• a second method is to use Liquid Chromatography (LC) to separate the proteins before being subjected to enzymatic hydrolysis (usually trypsin). The peptides (hydrolyzate) are then introduced into the mass spectrometer. These two techniques can advantageously be combined.
• Liquid chromatography used in combination with a mass spectrometer differs from High Performance Liquid Chromatography (HPLC for
• Performance Liquid Chromatography more generally used, mainly by the dimensions of the columns used and therefore by their flow rates.
• the diameter of the columns used is usually lmm (in contrast with average diameters of 4.6 mm for HPLC columns). More recently diameters of 300 .mu.m and even capillary type columns of diameters of 75 microns have become predominant.
• a particularly used source according to the invention is a "nanospray” source such as that marketed under the name Nanospray TM by the company Applied Biosystems, using Picotip emitter TM needles, SilicaTip, and coupled to an LC / mass spectrometer.
• MS / MS such as that marketed under the trademark Qstar XL TM T by Applied Biosystems.
• the mass spectrometry analysis used according to the invention comprises a separation step by liquid chromatography associated with tandem mass spectrometry (MS / MS).
• spots obtained by two-dimensional electrophoresis generally correspond to a single protein.
• the gel containing the stain is excised and subjected to enzymatic proteolysis.
• the most frequently used enzyme is trypsin.
• the peptide mass fingerprinting technique is used by which the masses of the peptides obtained are compared to a reference list on databanks which contains the list of the masses of the peptides. resulting from the digestion of known proteins. If the experimental data correspond to a given list, there is a high probability that the protein is present in the starting sample.
• Tandem analysis is particularly recommended in order to obtain information on the peptide sequence and thus to evaluate the relevance of the prior identification of the protein (validation). Tandem analysis involves isolating and then fragmenting the peptide ions in a collision chamber. In this method, the site of fragmentation is particularly localized to the peptide bonds. After fragmentation, the resulting mass spectra are compared by software to reference information contained in databases for protein identification.
• the peptides may be previously concentrated on a precolumn in an acid medium (C 18, pepMap 100, 5 ⁇ m, 100 ⁇ , 300 ⁇ m i.d.x 5 mm), mounted on a LC packing nano-chromatography chain (Dionex company).
• the peptides are then chromatographed on a C18 column, 3 ⁇ m, 75 ⁇ 150 mm, Atlantis TM (Waters company), the progressive elution of the peptides being carried out using a gradient (acetonitrile / water) coupled directly to the source of the spectrometer.
• massive. MS / MS analysis is particularly recommended for the identification of proteins from complex mixtures.
• biological marker denotes a biological molecule associated with the presence or absence of a particular pathological state, it is more particularly a protein.
• standard value is meant the average amount or concentration of biomarker present in healthy individuals.
• the method according to the invention can have various applications. Not only can it be useful for establishing a diagnosis or a prognosis but it can also be useful for the study or observation of the evolution of cutaneous pathologies or the effect of particular treatments as well as to make it possible to make a prognosis on the effectiveness of this or that treatment.
• this method is used to determine the existence of a positive response to the administration of a treatment. particular by identifying a significant variation in the presence of a particular marker. Such use makes it possible to more quickly determine the effectiveness of a particular treatment than to wait to visually observe a change in symptoms. It also reduces the costs associated with therapeutic studies of new drugs.
• the skin disease, or skin pathology, concerned by the method of the invention may be any cutaneous pathology. This is preferably an inflammatory and / or autoimmune or allergic disease.
• the cutaneous pathology is psoriasis and the marker to be identified is advantageously a marker of cutaneous psoriatic lesions.
• Psoriasis is a skin disease of poorly known origin, partly genetic that affects 2% of the population.
• the epidermis is renewed too quickly, in only four to six days, instead of the usual three weeks which generates localized inflammations.
• Epidermal cells accumulate on the surface of the skin and form a layer of white dandruff called scales. Perfectly harmless, they have the disadvantage of being unsightly.
• Psoriasis In its mild form, psoriasis is limited to the scalp, nails, knees, elbows, feet, hands and sometimes to the genitals. In severe cases, it extends and can gain the whole body. Psoriasis includes various symptomatic forms such as plaque psoriasis, psoriasis guttate, pustular psoriasis, reverse psoriasis, erythrodermic psoriasis and psoriatic arthritis. However, it is possible to control psoriasis, reduce the extent of the lesions and improve the lives of patients with certain treatments.
• the psoriasis marker that is detected or measured is selected from the group consisting of proteins having biological functions related to cell proliferation and differentiation.
• the laminin the skin-specific calmodulin-related protein (CLSP), the psoriasis-associated fatty acid binding protein, desmoplakin; psoriasin (SlOO A7) and alpha enolase, carcinoma cell antigen 1 and 2 (SCCA1, SCCA2).
• the psoriasis marker which is detected or measured is chosen as an example from the group consisting of markers having biological functions related to the response to stress and the immune and inflammatory response. .
• the heat shock proteins 27 (beta 1)
• the step of detecting a protein as a biological marker comprises identifying or determining the presence or absence of at least one protein.
• This step may also include determining the amount or concentration, relative or not, of said marker, and / or the variation of these values with respect to a given or standard value, as well as the evolution over time of these values.
• At least one skin sample for example a single sample, in particular of the upper layer of the epidermis (cornified stratum) is taken from a patient.
• the sample is taken at a location showing clinical signs of skin pathology, such as inflammation.
• the skin sample is taken from an inflamed part, it is preferred that the sample take place in the center of the plate so as to optimize the sampling.
• different areas of the lesion can be removed to determine certain characteristics (evolutionary zone, highly scaly zone, zone with strong erythema, treated or untreated zone).
• the sample is taken by affixing an adhesive to the patient's skin and then removing said adhesive to which a sample of stratum corneum adheres.
• This adhesive is chosen so that a sufficient skin sample is taken.
• This adhesive may advantageously consist of a translucent flexible plastic strip or not.
• the adhesive is preferably disposed on a support, such as a flexible support and preferably foldable so as to be able to best adapt to the contours and the elasticity of the part of the body on which it is applied, which makes it possible to obtain a skin sample, and more particularly a stratum corneum, also complete as possible.
• a support may consist for example of a plastic film.
• this adhesive may be pellets of D-squames TM (Monaderm), Sebutape type.
• Blenderm TM or ScotchTape TM (3M Company, St. Paul, Minn.), Or hydrogels such as those of the Hypan TM type (Hymedix International, Inc., Dayton, NJ.) , or any other type of material having adhesive properties such as glues, gums and resins.
• the adhesive is applied repeatedly to the skin until it becomes tacky.
• step a it is preferable to proceed on the cutaneous sample obtained in step a) to a step a ') of extraction of biological material.
• This extraction step can be carried out by any extraction protocol, especially protein extraction, which are well known to those skilled in the art.
• the invention thus particularly relates to a non-invasive method of collecting biological data useful for establishing a diagnosis or prognosis of a particular skin pathology in a patient, as previously described, in which the sample obtained in the sampling step a) is subjected to a biological material extraction step a '), then the detection step b) is performed on the extract thus obtained.
• the skin sample may advantageously be subjected to an enzymatic digestion step prior to analysis by mass spectrometry.
• This digestion step may advantageously be carried out by the use of trypsin.
• more this digestion step can be advantageously carried out in situ directly on the support of the adhesive which allows to increase the speed of analysis and reduce the amount of sample needed and thus allow to reduce the sampling area and / or to make more accurate sampling.
• the invention therefore particularly relates to a non-invasive method of collecting biological data useful for establishing a diagnosis or prognosis of a particular skin pathology in a patient as described above, in which the biological material contained in the cutaneous sample taken in step a) or obtained in step a ') is subjected to a step of enzymatic digestion a ") prior to analysis by mass spectrometry (step b)).
• the mass spectrometric analysis methods described above may be used, in particular spectral analysis in tandem MS / MS or spectrometry MALDI-TOF.
• the samples are placed in a tube (Eppendorf type) of 2 ml in the presence of a buffer solution (preferably ammonium bicarbonate pH 8.3) containing a reducing agent (preferably dithiothreitol) and a detergent compatible with the mass spectrometry analysis (preferably Rapigest TM, (Waters company)) for solubilizing the proteins and then an alkylating agent (preferably iodoacetamide) is added (step a ')).
• a buffer solution preferably ammonium bicarbonate pH 8.3
• a reducing agent preferably dithiothreitol
• a detergent compatible with the mass spectrometry analysis preferably Rapigest TM, (Waters company)
• an alkylating agent preferably iodoacetamide
• the peptides are directly loaded onto a pre-concentration (C 18) LC Packing column coupled to the mass spectrometer (step b).
• concentration is preferably carried out by a continuous gradient of water: acetonitrile mixture
• the peptides are separated in a column (Atlantis TM) and then ionized at the column outlet in the "nanospray" source for analysis in the mass spectrometer.
• the analysis of the mass spectra obtained is performed by a software allowing the interpretation of the mass spectra.
• Such software is, for example, MASCOT software (Matrix Science) Spectrum MiIl (Agilent company), Phenyx (Genebio company), ProID (Applied Biosystems company). These programs can be linked to databases such as UNIPROT, SWISSPROT or alternatively TrEMBL.
• step c) for separating the peptides before separation by nano-chromatography (step b) in order to better separate the peptides resulting from the enzymatic digestion.
• This step is particularly recommended for complex mixtures and for the search for proteins present in small quantities.
• This preliminary step may advantageously consist of a liquid chromatography (ion exchange) step.
• the peptides derived from an enzymatic digestion with trypsin are thus absorbed at acidic pH on an ion exchange column (anionic) and then eluted by a continuous gradient of salt of increasing concentration or by a succession of solutions containing increasing amounts of salt.
• Step The peptides thus eluted are directly (in line) absorbed on the pre-concentration column (C 18) and then chromatographed as described previously.
• the preliminary separation step c) may advantageously comprise a step of iso-focusing (or iso-electro-focusing) of the peptides on a gel comprising a immobiline gradient immobilized on the support (gel).
• This step is similar to the first protein separation step performed for two-dimensional protein analysis. The application of an electric current makes it possible to separate the peptides of different charges. The peptides after extraction of the gel are then directly chromatographed as indicated previously (nano-chromatography).
• the proteomic analysis is performed from a sample taken from the skin by applying a D-Squames TM type adhesive (Monaderm company).
• This product includes a flexible translucent polymer carrier and an adhesive.
• the application is repeated on the skin of the patient, preferably until the loss of adhesion.
• the sample obtained relates only to the content of the outermost layers of the epidermis (ie the
• the samples thus obtained are stored at -30 ° C. or -80 ° C. if storage for a longer period is desired.
• the sample obtained is incubated for 60 min in a 2 ml Eppendorf tube with constant stirring (at 1400 rpm) at 60 ° C. in 200 ⁇ l of a buffer solution of ammonium bicarbonate of pH 8.5 containing 0.1% RapiGest TM detergent (1 mg in 1000 ⁇ l) and 5 ⁇ M DTT (dithiothreitol). 15mM of iodoacetamine (IAA) (6 ⁇ l of a 0.5M solution) are then added and the mixture is incubated for 1h30 at room temperature in the dark.
• IAA iodoacetamine
• the sample is incubated with shaking (400 rpm) for 18 hours at 37 ° C. with 1 ⁇ l of a trypsin solution of porcine origin (1 mg / ml), then is acidified with 2% formic acid.
• the digestion product is filtered using 0.22 ⁇ m and 30 kDa filters under centrifugation (12000 rpm). The filtrate is used directly for analysis by liquid chromatography coupled with Qstar-XL mass spectrometry analytical mode (LC / MS / MS).
• Example 2 Determination of the presence of psoriasis markers that can be identified by non-invasive sampling of Stratum Corneum.
• Such markers can be advantageously used in a method according to the invention.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Urology & Nephrology (AREA)
• Immunology (AREA)
• Hematology (AREA)
• Physics & Mathematics (AREA)
• Cell Biology (AREA)
• Analytical Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Pathology (AREA)
• General Physics & Mathematics (AREA)
• Microbiology (AREA)
• Bioinformatics & Computational Biology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Spectroscopy & Molecular Physics (AREA)
• Biophysics (AREA)
• Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=39522122

Family Applications (1)

Country Status (5)

Families Citing this family (13)

Family Cites Families (6)

• 2007
  • 2007-11-14 FR FR0759039A patent/FR2923610B1/fr not_active Expired - Fee Related
• 2008
  • 2008-11-14 WO PCT/FR2008/052049 patent/WO2009068825A1/fr active Application Filing
  • 2008-11-14 EP EP08854335A patent/EP2223120A1/de not_active Ceased
  • 2008-11-14 CA CA2704613A patent/CA2704613A1/fr not_active Abandoned
• 2010
  • 2010-05-13 US US12/779,073 patent/US20100261215A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events