WO2017174557A2 - Microdialyse par traction/propulsion associée à une protéomique de dispersion pour analyser le protéome dans l'espace extracellulaire du cerveau - Google Patents

Microdialyse par traction/propulsion associée à une protéomique de dispersion pour analyser le protéome dans l'espace extracellulaire du cerveau Download PDF

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WO2017174557A2
WO2017174557A2 PCT/EP2017/057945 EP2017057945W WO2017174557A2 WO 2017174557 A2 WO2017174557 A2 WO 2017174557A2 EP 2017057945 W EP2017057945 W EP 2017057945W WO 2017174557 A2 WO2017174557 A2 WO 2017174557A2
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microdialysis
additive
perfusion liquid
sample
liquid
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PCT/EP2017/057945
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WO2017174557A3 (fr
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Thomas Ivo Franciscus Hubert Cremers
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Brains Online Holding B.V.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14507—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue specially adapted for measuring characteristics of body fluids other than blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14525—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using microdialysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6846—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive
    • A61B5/6847—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive mounted on an invasive device
    • A61B5/6866—Extracorporeal blood circuits, e.g. dialysis circuits

Definitions

  • the present disclosure relates generally to a method that comprises the use of sampling devices that enables sampling of brain extracellular space without limitation to size of molecules with concurrent proteomics analysis using mass spectrometry.
  • WO2011144914 describes a method of aiding the diagnosis of acute brain damage in a subject, said method comprising (i) assaying the concentration of at least one oxidative stress polypeptide selected from the group consisting of: PRDXl, PRDX6 and GSTP1 in a sample from said subject; and (ii) assaying the concentration of at least one further polypeptide selected from Panel A; (Hi) comparing the concentrations of (i) and (ii) to the concentrations of the polypeptides in a reference standard and determining quantitative ratios for said polypeptides; (iv) wherein a finding of a quantitative ratio of each of the assayed polypeptides in the sample to the polypeptides in the reference standard of greater than 1.3 indicates an increased likelihood of acute brain damage having occurred in said subject.
  • Microdialysis has been used for decades to sample extracellular space of brain and other compartments. This method is limited to the size of molecules extracted due to a minimal cut off of the membrane needed to prevent loss of dialysis fluid due to ultrafiltration. MS analysis of proteome has been used to study pathological processes and discover biomarkers in tissue, blood, CSF (Cerebrospinal fluid) and microdialysates.
  • the current invention describes the use of a high molecular weight cut off sampling technique in combination with MS proteomics, enabling study of full proteome of the extracellular space of the brain.
  • the method enables sampling of extracellular space without any transfer of fluid.
  • Microdialysis has been applied to measure neurotransmitters such and norepinephrine and dopamine, but also drugs in extracellular fluid.
  • the method allows sampling in many compartments such as brain, heart, skin, kidneys and bone of freely moving organisms.
  • Micro dialysis uses a semipermeable membrane to sample molecules of a low molecular weight.
  • a membrane contains pores of typically 5-30 KDalton and is perfused by a perfusion liquid (ringer or phosphate buffered saline, etc.).
  • perfusion liquid and “perfusion medium” may both be used and refer to the same type of liquid that is used for microdialysis.
  • the down side of microdialysis is that the pore size of the membrane will prevent larger molecules to be sampled. Increasing the pore size is not possible, as over a pore size of 30 KDalton, perfusion fluid will leak out of the membrane and damage surrounding tissue leading to artefacts in measurements. Whereas this is not detrimental when studying matrices like skin, in fragile matrices like brain it will typically lead to brain damage, convulsions and failed experiments. Previous methods that were investigated to sample brain without membranes, such as push pull, were prone to technical failures.
  • Microdialysis samples extracellular space are typically very low in abundance. Additionally, as perfusion rates are typically 0.2- 1.5 micro 1/min, typical 15 minute sample volumes are 3-22.5 microliter. Due to low concentrations and low volumes, dialysates allow little pre-concentration and sample preparation without technical problem such as recovery and loss of analyte.
  • Mass spectrometry coupled to liquid chromatrography in turn has been applied in multiple configurations such as HPLC coupled to Trap and TOF mass spectrometers, such as Orbitrap, Triple TOF 5600s or FT mass spectrometry.
  • Targeted mass spectrometry is also increasingly applied over the last decade. Multiple reaction monitoring has been shown to be useful in targeted mass spectrometry to attain better specificity and quantification.
  • Shotgun proteomics enables the simultaneous analysis of multiple peptides in a single run.
  • the technique is widely applied in a plethora of matrices such as plasma, serum, CSF and tissue homogenates.
  • the method has been applied on microdialysates to study proteomics of extracellular space of tumors, skin and brain.
  • the crucial step in the workflow of proteomics approaches is sample preparation. Removal of high abundance proteins from the matrix prior to analysis is of utmost importance for maximization of detection and quantification of mid and low abundance proteins.
  • Multiple separation methods can be distinguished, but typically methods are divided into enrichment methods that aim to enrich a subclass of proteins or peptides and chemical fractionation approaches, where proteins or peptides are separated based on their physical characteristics, such as charge, isoelectric point or size.
  • Multiple approaches have been suggested over the last decades, ranging from immuno-affinity separation (antibody based separation), protein size fractionation (gel based separation), chromatographic separation. Further sample preparation like digestion of peptides prior to analysis, is frequently employed in standard proteomics workflow to optimize the detection and quantification of peptides.
  • a first aspect of the invention provides a specific choice of membrane with sampling system that allows large molecules to traverse without size exclusion. Furthermore, as certain materials are prone to non-specific binding of apolair compounds like (certain) peptides, the present invention also provides details of materials of membranes and sampling system.
  • the invention provides in an aspect a microdialysis setup, especially suitable for push-pull microdialysis, comprising a microdialysis probe, the microdialysis probe comprising a first opening for introduction of a perfusion liquid, a second opening for removal of the perfusion liquid, and a membrane, wherein the microdialysis setup is configured for perfusion of perfusion liquid along one side of the membrane for retrieval of an analyte (from a liquid) at another side of the membrane, and wherein especially the membrane has a cut off equal to or larger than 0.1 MDa, especially in the range of 0.1-10 MDa, such as in the range of 0.2-10 MDa.
  • such microdialysis setup may further comprise a first conduit for transport of the perfusion liquid to the microdialysis probe, wherein the first opening is in liquid contact with the first conduit.
  • such microdialysis setup may further comprise a second conduit for transport of the perfusion liquid from the microdialysis probe to a sample receiver, wherein the second opening is in liquid contact with the second conduit.
  • such microdialysis setup may further comprise a liquid transport system comprising one or more of (a) a first conduit for transport of the perfusion liquid to the microdialysis probe, wherein the first opening is in liquid contact with the first conduit, and (b) a second conduit for transport of the perfusion liquid from the microdialysis probe to a sample receiver, wherein the second opening is in liquid contact with the second conduit.
  • a liquid transport system comprising one or more of (a) a first conduit for transport of the perfusion liquid to the microdialysis probe, wherein the first opening is in liquid contact with the first conduit, and (b) a second conduit for transport of the perfusion liquid from the microdialysis probe to a sample receiver, wherein the second opening is in liquid contact with the second conduit.
  • such microdialysis setup may further comprise a peristaltic pump for flowing the perfusion liquid through the liquid transport system.
  • the peristaltic pump is configured downstream of the probe.
  • the setup is configured to provide a negative pressure to the second conduit and/or to use gravitational pull to an outlet of the second conduit (especially for a gravitational pull on the outlet fluid of the microdialysis setup).
  • such microdialysis setup may further comprise a syringe pump configured upstream of the probe, wherein especially the syringe pump is in liquid contact with an inlet of the first conduit, and wherein an outlet of the first conduit is in liquid contact with the first opening of the microdialysis probe.
  • such microdialysis setup may further be configured to flow the perfusion liquid along one side of the membrane for with a flow rate selected from the range of 0.1-2 ⁇ /min.
  • such microdialysis setup may further comprise one or more of the first conduit, the second conduit, and the sample receiver comprise a material selected from the group consisting of PEEK (Polyether ether ketone), FEP (fluorinated ethylene propylene), polyethylene, and polypropylene.
  • PEEK Polyether ether ketone
  • FEP fluorinated ethylene propylene
  • polyethylene polyethylene
  • polypropylene polypropylene.
  • the membrane comprises one or more of poly ether sulfone (PES), polyethylene, poly aryl ether sulfone (PAES), and polyether (PE).
  • the second conduit may comprise one or more of Tygon or silicone.
  • Tygon may comprise one or more of Plastic, Silicone, PVC, Polyurethane, Fluoropolymer, Thermoplastic Elastomer and Plasticizer Free; like one or more of Silicone, PVC, Polyurethane, Fluoropolymer, Thermoplastic Elastomer.
  • such microdialysis setup may further comprise a temperature control element configured to control the temperature of a sample receiver, wherein the temperature control element is especially configured to cool the sample receiver to a temperature selected from the range of 1 - 10 °C.
  • such microdialysis setup may further comprise a reservoir for an additive for the perfusion liquid.
  • the reservoir for an additive for the perfusion liquid is configured upstream of a reservoir for the perfusion liquid.
  • the reservoir for an additive for the perfusion liquid is configured in liquid contact with a first conduit for transport of the perfusion liquid to the microdialysis probe, and wherein an outlet of the reservoir for an additive for the perfusion liquid is configured downstream from an outlet of a reservoir for the perfusion liquid.
  • the perfusion liquid further comprises one or more of albumin and glycerol.
  • such additive like one or more of albumin and glycerol, are added to the perfusion liquid.
  • the microdialysis setup is configured to provide the additive to the perfusion liquid downstream from the membrane, such as for providing a protease inhibitor additive.
  • the microdialysis setup is configured to provide an additive to the perfusion liquid, especially downstream from the membrane, wherein such additive may limit or prevent non-specific binding.
  • such additive may comprise one or more of albumin and glycerol.
  • the microdialysis setup is configured to provide one or more additive to the perfusion liquid upstream of the membrane and configured to provide one or more additive to the perfusion liquid downstream of the membrane.
  • the invention also provides an arrangement of apparatus comprising the microdialysis setup as defined herein and one or more of a LC-MSMS apparatus and a refrigerator for storing a microdialysis sample at a temperature equal to or below 0 °C, especially equally to or below -20 °C, such as equal to or below liquid nitrogen temperature.
  • the invention also provides a method of providing a perfusion liquid, the method comprising providing a perfusion liquid with standard perfusion liquid materials and an additive, wherein the perfusion liquid comprises water, wherein the standard perfusion liquid materials (in addition to water) further comprises one or more of sodium, potassium, magnesium, calcium, and optionally one or more of phosphate and lactate, and wherein the additive comprises one or more of (i) an additive configured to inhibit non-specific binding of a protein, such as one or more selected from the group consisting of albumin and cyclodextrin, (ii) a degradation inhibitor, such as one or more selected from the group consisting of a protease inhibitor, (iii) a preservative selected from the group consisting of formic acid, and ascorbic acid, and (iv) an additive to prevent or reduce non-specific binding, such as especially one or more of glycerol, DMSO and methanol.
  • the perfusion liquid comprises water
  • the standard perfusion liquid materials in addition to water
  • the additive comprises
  • such method may further comprise combining the standard perfusion liquid materials to provide the perfusion liquid and mixing the perfusion liquid with the additive.
  • such method may further comprise combining the standard perfusion liquid materials and one or more additives to provide the perfusion liquid comprising said additive.
  • the invention also provides a perfusion liquid comprising standard perfusion liquid materials and an additive, wherein the perfusion liquid comprises water, wherein the standard perfusion liquid materials further comprises one or more of sodium, potassium, magnesium, calcium, and optionally phosphate, and wherein the additive comprises one or more of (i) an additive configured to inhibit non-specific binding of a protein, such as one or more selected from the group consisting of albumin and cyclodextrin, (ii) a degradation inhibitor, such as one or more selected from the group consisting of a protease inhibitor, (iii) a preservative selected from the group consisting of formic acid, and ascorbic acid, and (iv) an additive to prevent or reduce non-specific binding, such as especially one or more of Glycerol, DMSO and methanol.
  • the invention also provides a method of providing a perfusion liquid as defined herein to the microdialysis setup as defined herein, the method comprising providing said standard perfusion liquid
  • such method may further comprise providing the standard perfusion liquid materials to a reservoir for the perfusion liquid and providing the additive to a reservoir for the additive for the perfusion liquid.
  • such method may further comprise providing the combination of the standard perfusion liquid materials and the additive for the perfusion liquid to a reservoir.
  • the invention provides in yet a further aspect a method for protecting of a microdialysis sample, the method comprising providing an additive to the microdialysis sample, wherein the sample is optionally contained in a sample receiver, wherein the method comprises adding an additive to the microdialysis sample, and optionally mixing the additive and microdialysis sample, wherein the additive comprises one or more of (i) an additive configured to inhibit non-specific binding of a protein, such as one or more selected from the group consisting of albumin and cyclodextrin, (ii) a degradation inhibitor, such as one or more selected from the group consisting of a protease inhibitor, (iii) a preservative selected from the group consisting of formic acid, and ascorbic acid, and (iv) an additive to prevent or reduce non-specific binding, such as especially one or more of Glycerol, DMSO and methanol.
  • the additive comprises one or more of (i) an additive configured to inhibit non-specific binding of a protein, such as
  • the microdialysis sample comprises brain extracellular fluid, and wherein especially the sample receiver comprise a material selected from the group consisting of PEEK (polyether ether ketone), FEP (fluorinated ethylene propylene), polyethylene, and polypropylene.
  • PEEK polyether ether ketone
  • FEP fluorinated ethylene propylene
  • the invention provides a method of analyzing a microdialysis sample, wherein especially the microdialysis sample is an ex vivo sample, wherein the microdialysis sample comprises an additive comprising one or more of (i) an additive configured to inhibit non-specific binding of a protein, such as one or more selected from the group consisting of albumin and cyclodextrin, (ii) a degradation inhibitor, such as one or more selected from the group consisting of a protease inhibitor, (iii) a preservative selected from the group consisting of formic acid, and ascorbic acid, and (iv) an additive to prevent or reduce non-specific binding, such as especially one or more of Glycerol, DMSO and methanol, and wherein the method comprises subjecting the microdialysis sample to LC- MSMS analysis.
  • an additive configured to inhibit non-specific binding of a protein, such as one or more selected from the group consisting of albumin and cyclodextrin
  • a degradation inhibitor such
  • such method may further comprise subjecting the microdialysis sample to HPLC-MSMS analysis, and/or wherein the method comprises prior to LC-MSMS analysis one or more or a precipitation of the sample and a digestion of the sample.
  • the microdialysis sample comprises brain extracellular fluid, especially obtainable by push-pull microdialysis.
  • other bodily material such as other bodily fluids or tissue may also be possible.
  • the microdialysis sample may also be a tumor or a tumorous tissue.
  • the bodily fluid may be (investigated) ex vivo or in vivo.
  • the invention also provides a method where brain extracellular fluid sampling is performed by a method that has limited size exclusion and is combined with shotgun proteomics for analysis, especially identification of new targets and/or pathophysiology of the brain.
  • such method may be used where sampling is performed using push pull microdialysis.
  • push pull microdialysis is performed using a membrane with a cut off larger than 0.1 mega Dalton, especially larger than 0.2 mega Dalton, and wherein especially the membrane comprises one or more of poly ether sulfone (PES), polyethylene, poly aryl ether sulfone (PAES), and polyether (PE).
  • PES poly ether sulfone
  • PAES polyaryl ether sulfone
  • PE polyether
  • sampling is performed using push pull microdialysis, push pull dialysis, open flow dialysis or ultrafiltration.
  • perfusion of the microdialysis sample is performed with ringer or PBS containing albumin.
  • perfusion of the sample is performed with ringer or PBS containing a cyclodextrine.
  • a preservative is added post dialysis; optionally, also a compound may be added that may limit or prevent non-specific binding (such as albumin or glycerol).
  • analysis of a sample is performed using shotgun proteomics.
  • the sample is digested prior to analysis, especially wherein a protease, such as trypsin, is added to the sample.
  • a protease such as trypsin
  • the sample is chemically precipitated, especially with acetonitrile, especially prior to digestion. Acetonitrile may be used to limit or prevent nonspecific binding.
  • the additive at least comprises one or more of glycerol and albumin. Therefore, in embodiments the additive of the perfusion liquid at least comprises one or more of glycerol and albumin. With such additive(s) the recovery may be better. It seems that non-specific binding may be reduced when one or more of glycerol and albumin are applied, especially when both are applied. Further, the present invention provides a specific use of materials for sampling. As the microdialysis probe is connected to tubings that will transfer the dialysate to the collection vials, the materials of these tubings should not be prone to (non-specific) binding of analytes. In an embodiment of the invention PEEK (polyether ether ketone) or FEP (fluorinated ethylene propylene) tubings are used.
  • PEEK polyether ether ketone
  • FEP fluorinated ethylene propylene
  • Dialysis methods like push pull microdialysis and open flow dialysis in brain need a negative pressure on the outlet (second opening or conduit opening of conduit in liquid contact with this outlet) in order to pull the dialysate out and prevent ultrafiltration in the brain.
  • the sample is pulled out by negative pressure that is induced by placing the outlet of the dialysis system (herein also indicated "microdialysis setup") under the sampling position.
  • microdialysis setup the outlet of the dialysis system
  • Another method to provide negative pressure is to place the sample collection system under negative pressure. Both methods prevent the use of pumping systems which require materials such as silicone tubing or other materials prone to non-specific binding.
  • the pressure may e.g. in the range of 0.5-0.99 bar, like 0.8-0.95 bar.
  • a peristaltic pump is used to pull the sample out. Optimal conditions to prevent non-specific binding of analytes in the system are described.
  • albumin may in embodiments be added to provide an amount of about 0.1-1.5 wt.%, especially 0.25-1.0 wt.%, such as about 0.5 wt.%. In embodiments, albumin may be added to provide an amount of about 0.1-6 wt.%, especially 0.20-5.0 wt.%, such as about 4 wt.%>.
  • Cyclodextrin may be added to provide an amount of about 0.5-5.0 wt.%), especially 1.0-4.0 wt.%>, such as about 0.5 wt.%>. Amounts are relative to the total amount of perfusion liquid.
  • additive may also refer to a plurality of different additives.
  • Pre-conditioning the sampling system is important in order to prevent loss of analytes. Preconditioning of tubings by flushing overnight with albumin further helps loss of analyte during sampling.
  • a further embodiment of the invention uses the inclusion of degradation inhibitors such as protease inhibitors in the dialysis medium or in the collection vial.
  • a post dialysis make up flow might be used to add protease inhibitor online during sampling, though optionally the protease inhibitor may also be added to the perfusion liquid (upstream of the microdialysis probe).
  • polypropylene or poly ethylene 300 ⁇ vials are used in a sample collector at 4 °C. Samples are immediately frozen at -80 °C upon collection.
  • preservatives are added to the dialysis fluid to prevent degradation of analytes.
  • Preservatives can alter pH such as formic acid, prevent oxidation such as ascorbic acid or prevent enzymatic degradation such as protease inhibitors.
  • Solvents that prevent non-specific binding such as DMSO or glycerol and modifiers such as methanol or acetonitrile might also be used.
  • Sample preparation is beneficial to prepare the sample for adequate analysis. Removal of high abundance proteins, lipids and other molecules might be performed using precipitation with acetonitrile, size exclusion chromatography, immunnoaffmity chromatography or SDS page (sodium dodecyl sulfate polyacrylamide gel electrophoresis).
  • Proteins might be digested prior to analysis using a protease, such as trypsin. Digestion might be performed in the samples, in gel after separation, or online during chromatography.
  • a protease such as trypsin.
  • Concentrations of analytes of interest might be very low in extracellular fluid.
  • Fig la shows the schematic setup of the microdialysis experiment.
  • Push pull microdialysis probes are connected to a syringe pump using peek tubing and perfused with ringer containing albumin at a flow of 0.5 micro 1/min.
  • Solution containing preservatives like protease inhibitors and/or or compounds that prevent non-specific binding like albumin or glycerol can be added dialysis.
  • Samples, herein also indicated as "microdialysis samples” are collected using gravitational pull and collected in 300 micro 1 microdialysis vial;
  • Fig lb is a schematic representation of the dialysis sampling system.
  • Perfusion medium is infused through the inlet and runs back to the outlet through the dialysis membrane. Peptides will traverse over the membrane and enter the perfusion medium.
  • the flow Before leaving the microdialysis probe, the flow enters a low volume mixing room, were a make-up flow (or additional flow) containing e.g. a protease inhibitor and compounds that prevent non-specific binding like albumin and glycerol are added.
  • the dialysate leaves the microdialysis probe via the outlet; and
  • Fig. 2 shows the schematic workflow of the sampling, sample preparation and analysis of the experiment.
  • Fig. 3 shows the relative recovery of amyloid beta 1-40 using different push pull microdialysis techniques. Recovery was Metaquant push pull microdialysis»gravitational push pull dialysis»conventional push pull dialysis.
  • mice were anesthetized using isoflurane (2%, 800 mL/min 02). Bupivacain/epinephrine was used for local analgesia. The analgesic Finadyne was used for peri-/post-operative analgesia. The animals were placed in a stereotaxic frame (Kopf instruments, USA) and push pull microdialysis probes (3 mm exposed surface, FractioPES 200 membrane; Brainlink, the Netherlands) were inserted into the striatum.
  • the toothbar was set at 0.0 mm (Paxinos and Franklin, 2004). After surgery, animals were kept individually in cages, provided food and water ad libitum.
  • Microdialysis experiments Experiments were performed one day after surgery. On the day of the experiment, the probes of the animals were connected with flexible PEEK tubing to a microperfusion pump (Harvard PHD 2000 Syringe pump, Holliston, MA or similar). The push pull microdialysis probes were perfused with 0.2% BSA in aCSF (artificial cerebrospinal fluid) containing 147 mM NaCl, 3.0 mM KC1, 1.2 mM CaCl 2 and 1.2 mM MgCl 2 , at a flow rate of 0.5 ⁇ / ⁇ .
  • aCSF artificial cerebrospinal fluid
  • Microdialysis samples were collected for 60-minute periods by an automated fraction collector (820 Microsampler, Univentor, Malta) into polypropylene mini- vials already containing 10 of 40% glycerol + 0.2% BSA in aCSF. The final total sample volume was 40 ⁇ . The microdialysate samples were stored at -80 °C awaiting their analysis.
  • the gel band was sliced into small pieces, washed subsequently with 30% and 50% v/v acetonitrile with 100 mM ammonium bicarbonate, each incubated at RT for 30 min while mixing (500 rpm) and lastly with 100% acetonitrile for 5 min.
  • the gel pieces were dried in an oven at 37 °C before overnight digestion with 20 ⁇ , trypsin (1 : 100 g/g, sequencing grade modified trypsin V5111, Promega) at 37 °C.
  • Peptide mixtures were loaded onto a trapping microcolumn (Acclaim PepMap; CI 8; 5-mm length by 300- ⁇ inside diameter; 5- ⁇ particle size; 100-A porosity; Dionex) in 0.1% formic acid at a flow rate of 20 ⁇ / ⁇ .
  • a trapping microcolumn Acclaim PepMap; CI 8; 5-mm length by 300- ⁇ inside diameter; 5- ⁇ particle size; 100-A porosity; Dionex
  • peptides were eluted onto a nanocolumn (50 cm x 75 ⁇ ) packed with CI 8 PepMAPlOO 3 ⁇ particles (Dionex).
  • the following mobile-phase gradient was delivered at a flow rate of 250 nL/min: 2-50% solvent B for 60 min, 50 to 90% B for 7 min, 90% B for 10 min, and back to 2% B for 5 min.
  • Solvent A was water + 0.1% formic acid, and solvent B acetonitrile + 0.1% formic acid.
  • Peptides were infused with a stainless steel emitter (Proxeon, Odense, Denmark) at a typical spray voltage of 1.8 kV with no sheath and auxiliary gas flow; the ion transfer tube temperature was 200°C.
  • Data-dependent acquisition (DDA) cycle was performed with a survey scan of m/z 400 to 1800 at a target mass resolution of 70,000, followed by MS/MS fragmentation of the eight most intense precursor ions. Singly charged ions were excluded from MS/MS experiments, and the m/z values of fragmented precursor ions were dynamically excluded for a further 60 s.
  • PEAKS Studio software version 7.5 was used to search the LC-MS/MS data against the mouse reference proteome (UniProt) at a parent mass error tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da.
  • the false discovery rate (FDR) was set at 0.1%.
  • Table 1 shows a list of peptides detected in typical dialysates from a push pull experiment according to the invention, with table la showing the protein ID, accession, - lOlgP, and coverage (%) for the protein groups and with table lb showing for the same protein groups the number of peptides (#peptides), the number of unique PTMs (post- translational modifications) (#Unique PTM), the average mass (Avg. Mass), and a description.
  • Table 2 shows the relative abundance of peptides quantified in different age groups, normalized on total ion current of the chromatographic run. Relative abundance is 1/16 at -, 1/4 at -, 1 ⁇ 2 at -, 0 at 0, 2 at +, 4 at ++ and 16 at +++.
  • OS Mus musculus Protein #Peptides #Unique PTM Avg. Description
  • Probes were placed in stirred human serum at 37°C.
  • the push pull microdialysis probes were perfused with 0.2% BSA in aCSF (artificial cerebrospinal fluid) containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl 2 and 1.2 mM MgCl 2 , at a flow rate of 0.5 ⁇ / ⁇ .
  • aCSF artificial cerebrospinal fluid
  • Metaquant push pull microdialysis probes were perfused with 0.2%> BSA in aCSF (artificial cerebrospinal fluid) containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaC12 and 1.2 mM MgC12, at a flow rate of 0.5 ⁇ / ⁇ ⁇ using a syringe pump (CMA 102).
  • CMA 102 A push pull microdialysis probe of the metaquant design (fig 1A and 1 B) was used for these experiments.
  • a second syringe pump (CMA 102) was used to deliver a post dialysis make up flow of 3.8% BSA and 20% glycerol at a flow rate of 0.5 ⁇ / ⁇ .
  • a peristaltic pump (tygon tubing 0.12 mm) was used to pull the sample out of the probe at 1 ⁇ / ⁇ .
  • the inlet tubing was PEEK, and the outlet tubing was FEB with 20 cm of ty
  • a metaquant push pull microdialysis probe may especially be configured according to figures 1 a and lb, were a probe contains an inlet for infusion of perfusion medium. After perfusion of the dialysis membrane, the microdialysis probe comprises a mixing room that contains an inlet for a make-up flow (containing compounds that prevent degradation (for instance protease inhibitors), non-specific binding (for instance albumin and glycerol)) and an outlet for transfer of the mixed dialysis fluid and make up flow to a sample vial with the help of a peristaltic pump or gravitational pull.
  • a make-up flow containing compounds that prevent degradation (for instance protease inhibitors), non-specific binding (for instance albumin and glycerol)
  • an outlet for transfer of the mixed dialysis fluid and make up flow to a sample vial with the help of a peristaltic pump or gravitational pull for example, albumin and glycerol
  • Microdialysis samples were collected for 30-minute periods by an automated fraction collector (820 Microsampler, Univentor, Malta) into polypropylene mini-vials. After an initial stabilization period of 60 minutes, 30 min dialysis samples were collected for analysis.
  • an automated fraction collector 820 Microsampler, Univentor, Malta
  • Shotgun proteomics analysis In order to purify the sample, 50 pL of dialysis sample was mixed in SDS loading buffer (0.01% bromophenol blue, 2% SDS, 4% glycerol, 1% ⁇ -mercaptoethanol in 150 mM Tris buffer (pH 6.8)). The sample was run briefly into a precast 4-12% Bis-Tris gels (Novex, run for maximally 5 min at 100 V). The gel was stained with Biosafe Coomassie G-250 stain (Biorad) and after destaining with milliQ, the band containing all proteins was excised from gel.
  • SDS loading buffer 0.01% bromophenol blue, 2% SDS, 4% glycerol, 1% ⁇ -mercaptoethanol in 150 mM Tris buffer (pH 6.8).
  • SDS loading buffer 0.01% bromophenol blue, 2% SDS, 4% glycerol, 1% ⁇ -mercaptoethanol in 150 mM Tris buffer (pH 6.8).
  • the gel band was sliced into small pieces, washed subsequently with 30% and 50% v/v acetonitrile with 100 mM ammonium bicarbonate, each incubated at RT for 30 min while mixing (500 rpm) and lastly with 100% acetonitrile for 5 min.
  • the gel pieces were dried in an oven at 37 °C before overnight digestion with 20 pL trypsin (1 : 100 g/g, sequencing grade modified trypsin V5111 , Promega) at 37 °C.
  • Peptide mixtures were loaded onto a trapping microcolumn (Acclaim PepMap; CI 8; 5-mm length by 300- ⁇ inside diameter; 5- ⁇ particle size; 100-A porosity; Dionex) in 0.1% formic acid at a flow rate of 20 ⁇ / ⁇ .
  • a trapping microcolumn Acclaim PepMap; CI 8; 5-mm length by 300- ⁇ inside diameter; 5- ⁇ particle size; 100-A porosity; Dionex
  • peptides were eluted onto a nanocolumn (50 cm x 75 ⁇ ) packed with CI 8 PepMAPlOO 3 ⁇ particles (Dionex).
  • the following mobile-phase gradient was delivered at a flow rate of 250 nL/min: 2-50% solvent B for 60 min, 50 to 90% B for 7 min, 90% B for 10 min, and back to 2% B for 5 min.
  • Solvent A was water + 0.1% formic acid, and solvent B acetonitrile + 0.1% formic acid.
  • Peptides were infused with a stainless steel emitter (Proxeon, Odense, Denmark) at a typical spray voltage of 1.8 kV with no sheath and auxiliary gas flow; the ion transfer tube temperature was 200°C.
  • Data-dependent acquisition (DDA) cycle was performed with a survey scan of m/z 400 to 1800 at a target mass resolution of 70,000, followed by MS/MS fragmentation of the eight most intense precursor ions. Singly charged ions were excluded from MS/MS experiments, and the m/z values of fragmented precursor ions were dynamically excluded for a further 60 s.
  • PEAKS Studio software version 7.5 was used to search the LC-MS/MS data against the mouse reference proteome (UniProt) at a parent mass error tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da.
  • the false discovery rate (FDR) was set at 0.1%.
  • Table 5 shows the amount of proteins identified in samples of different push pull microdialysis techniques. Amount of proteins was Metaquant push pull microdialysis>Gravitational pull microdialysis>conventional pull microdialysis. Table 4. comparison of protein intensity between metaquant push pull microdialysis, conventional push pull microdialysis and gravitational push pull microdialysis:
  • Table 5 shows the amount of proteins identified in samples of different push pull microdialysis techniques:

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Abstract

L'invention comprend un procédé dans lequel l'espace extracellulaire est échantillonné en utilisant des techniques de dialyse à point de coupure élevé en association avec une protéomique utilisant l'analyse LC-MS/MS après précipitation et digestion d'échantillons.
PCT/EP2017/057945 2016-04-04 2017-04-04 Microdialyse par traction/propulsion associée à une protéomique de dispersion pour analyser le protéome dans l'espace extracellulaire du cerveau WO2017174557A2 (fr)

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