WO2012152943A1 - Composés et procédés pour l'identification et/ou la quantification de biomolécules carbonylées - Google Patents

Composés et procédés pour l'identification et/ou la quantification de biomolécules carbonylées Download PDF

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WO2012152943A1
WO2012152943A1 PCT/EP2012/058844 EP2012058844W WO2012152943A1 WO 2012152943 A1 WO2012152943 A1 WO 2012152943A1 EP 2012058844 W EP2012058844 W EP 2012058844W WO 2012152943 A1 WO2012152943 A1 WO 2012152943A1
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compound
paca
carbonylated
compounds
biomolecules
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Lina SELLAMI
Claude VILLARD
Jean Michel Brunel
Daniel LAFITTE
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Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
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Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/62—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
    • 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
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the subject invention provides compounds and methods for derivatizing, identifying and/or quantifying carbonylated bio molecules by using compounds of the invention.
  • Oxidative stress is thought to be involved in many disorders ranging from neuronal to metabolic to chronic inflammatory diseases. It is widely accepted that reactive oxygen species (ROS) are responsible for damaging biomolecules, such as lipids, sugars, DNA and, in particular, proteins. ROS are also involved in in vitro processes in many other fields and processes, such as in the food industry, where oxidation of polyunsaturated fatty acids and proteins present problems in the production, storage and aging of foodstuffs. It is known that protein carbonylation arises from oxidative attack, and is therefore the major indicator of oxidative stress in biological samples. Carbonyl derivatives of proteins are formed by a direct attack of reactive oxygen species on specific amino acid side chains or can be the result of secondary reactions with reactive carbonyl compounds of advanced glycation or lipoxidation end products.
  • Determining the identity of proteins susceptible to carbonylation in vitro and in vivo can provide a new level of information that may be critical to understanding the specific pathophysiological consequences of oxidative stress-induced damage.
  • Oxidative stress is conventionally analyzed using various derivatization and detection methods, for example derivatization with 2,4-DNPH (i.e., 2,4-dinitrophenylhydrazine), and detection with antibodies directed against the DNP (i.e., dinitrophenyl) moiety.
  • 2,4-DNPH i.e., 2,4-dinitrophenylhydrazine
  • detection with antibodies directed against the DNP i.e., dinitrophenyl
  • the major current derivatization method is with biotin hydrazide, and this has been recently employed in conjunction with detection by avidin-coupled techniques by Hensley et al., 2009. Fluorescence based methods are also in effect.
  • Carbonyl trapping mechanisms using a hydrazide moiety are known in the art, for example US 2001/0036538 and US 2005/0250152.
  • the present invention provides a highly specific marker for the detection/measurement of carbonylated biomolecules, which can provide information that may be critical to understanding the specific pathophysiological consequences of oxidative stress-induced damage.
  • the method of the invention by using the presently disclosed compounds offers the advantage of simultaneous derivatization, quantification and enrichment of carbonylated biomolecules, which is fully compatible with analysis by mass spectrometry.
  • the invention provides compounds and methods for enriching, identifying and/or quantifying carbonylated biomolecules by using compounds of the invention.
  • the current invention thus provides compounds and a method of using such compounds for the analysis of carbonylated bio molecular species (such as peptides, proteins, and lipids).
  • This method advantageously allows simultaneous derivatizing and enrichment of carbonylated samples and is fully amenable to analysis by mass spectrometry, in particular in MS and MS/MS mode.
  • Mass spectrometry-based methods have in addition the potential to determine the localization of protein carbonylation.
  • proteins, or more generally biomolecules, that give rise to a reactive carbonyl upon oxidation are identified using the compounds and methods of the invention.
  • proteins, or more generally biomolecules, that give rise to a reactive carbonyl upon oxidation are identified using the compounds and methods of the invention.
  • one object of the invention is the identification and usage of protein carbonyl groups as biomarkers of oxidative stress.
  • the present invention relates to a method of the invention comprising the following steps: (i) biomolecules derivatization by the compound of the invention, (ii) identification of specifically carbonylated biomolecules, (iii) optional quantification of carbonylated species and/or of the rate of carbonylation.
  • the term “derivatization” refers to the step where the carbonyl groups of the biomolecules are reacted with the compounds of the invention.
  • identification refers to the determination of the nature (for instance for lipids) and/or sequence (for instance for peptides, proteins, or the like) of the carbonylated biomolecules.
  • the identification step may further include the localization of the carbonylation on the biomolecule.
  • the identification step can be more specifically carried out by using spectrophotometry and/or mass spectrometry techniques.
  • Quantification refers to the determination of the proportion of carbonylated biomolecules in the sample and/or of the number of carbonylations per biomolecule.
  • biomolecular species and “biomolecule” refer to a single biomolecule or to a mixture of biomolecules, in particular peptides, proteins, lipids, and the like. These terms may also refer to a biological sample comprising at least one biomolecule susceptible to carbonylation.
  • the present invention relates to compounds having the general formula (I):
  • Ri, R 2 , R 3 , and R 4 identical or different, represent H, OH, NH 2 , (C l-C8)alkyl, (C3-C8)cycloalkyl, (C6-C18)aryl, heteroaryl, heterocycle, halogen, or a silyl group, such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups.
  • m is 0 and at least one of Ri, R 2 , R 3 , and R 4 represents NH 2 , (C2-C8)alkyl, (C3-C8)cycloalkyl, (C6-C18)aryl, heteroaryl, heterocycle, or a silyl group as defined above.
  • the present invention more specifically discloses compounds having the general formula 1 , 2, 3, 4, 5 or 6:
  • n, R l s R 2 , R3, and R4 are as defined above, m is 0 for compounds 1 , 2 and 3. m is 1 for compounds 4, 5 and 6.
  • a more specific compound of the invention is a compound of formula 4 wherein R l s R 2 , R3, and R4 are hydrogen atoms and n is 2.
  • n is equal to 1 , 2, 3, 4, 5, or 6.
  • a highly preferred value for n is 4.
  • alkyl when used alone or in combination with other terms, comprises an optionally substituted straight or branched Ci-Cs alkyl chain which refers to monovalent alkyl groups having 1 to 8, preferably 2 to 8, carbon atoms.
  • This term is exemplified by groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 1-ethylpropyl, 2- methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, n-hexyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, n- heptyl, n-octyl.
  • the alkyl group has 1 to 4 carbon atoms.
  • cycloalkyl when used alone or in combination with other terms, refers to an optionally substituted saturated carbocycle of from 3 to 8 carbon atoms having a single ring (e.g cyclohexyl) or multiple condensed rings (e.g norbornyl).
  • aryl when used alone or in combination with other terms, refers to an optionally substituted unsaturated aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (e.g phenyl) or multiple condensed rings (e.g indenyl, naphthyl).
  • aryl includes phenyl, naphthyl, anthryl, phenanthryl and the like.
  • the aromatic group may be at least partially hydrogenated.
  • heterocycle refers to a cycloalkyl group as defined above, substituted in at least one position of the cycle with a heteroatom that may be an atom of N, O or S.
  • heteroaryl refers to an aryl group as defined above, substituted in at least one position of the cycle with a heteroatom that may be an atom of N, O or S.
  • halogen refers to any atom from group 17 of the "IUPAC" periodic table, comprising in particular chlorine, fluorine, bromine, or iodine.
  • silyl groups are well known in the art and can be selected by anyone of ordinary skill in the art, for instance, among alkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups.
  • the approach involves therefore the synthesis and use of compounds of formula (I), in particular of formulae (1) to (6) that are capable of providing these functions, having the generic name "PACa” (which stands for "Piege A Carbonyles”).
  • PACa which stands for "Piege A Carbonyles”
  • the compounds of the invention are suitable for both in vivo and in vitro studies. They allow derivatization and identification of the carbonylated species by spectrophotometry and/or mass spectrometry and specific enrichment of the carbonylated species, for instance in complex samples.
  • the compounds of the invention are thus brought into contact with a biomolecule, such as a peptide or lipid, in the derivatization step.
  • a biomolecule such as a peptide or lipid
  • the compounds of the invention are highly specific to reaction with carbonyls only and form stable hydrazones.
  • the compounds of the invention are small molecules, they are susceptible to react with several carbonyl groups in the same biomolecule, contrary to biotin hydrazide that is classically used for detection of carbonylated biomolecules.
  • Simultaneous reaction with several carbonylated groups of the same biomolecule affords the possibility to quantify the rate of carbonylation of said biomolecule, in other words the proportion of carbonylated aminoacids per biomolecule. Quantification of the rate of carbonylation of the biomolecule may be performed subsequently to contacting the biomolecule with a compound of the invention, as described below.
  • the compounds of the invention may be mounted on supports, either before or after derivatization with the biomolecule, more specifically via disulphide bonds through the thiol groups on the compounds of the invention.
  • the support on which the compounds of the invention may be mounted on gold supports, thio-silica magnetic beads, sepharose columns, and/or any other solid support susceptible to be reacted with the thiol function of the compounds of the invention, for instance susceptible to bear thiol groups that can be reacted with the thiol function of the compounds of the invention.
  • the compounds mounted on a support facilitate analyses, in particular they allow an enrichment step where the concentration of carbonylated species can be amplified.
  • enrichment refers to increase the concentration of the carbonylated biomolecular species linked to the compound of the invention, with respect to the various other species in a sample. This may be associated with a separation process, for instance via disulfide bonds reduction, whereby only the carbonylated species are collected for further analysis.
  • Quantification of the carbonyl content of the derivatized biomolecules may be carried out for instance by using UV/Vis spectroscopy or mass spectrometry.
  • mass spectrometry may be used both to confirm the derivatization of the biomolecule by the compounds of the invention did take place, and to identify the biomolecule.
  • Derivatization of the biomolecule by the compounds of the invention is assessed by the detection of a specific mass on the mass spectrometry spectra.
  • This specific mass is that of a "reporter ion” and corresponds to a fragment of compounds of the invention. Detection of this ion is a proof of the derivatization of the biomolecule with the compound of the invention.
  • this specific mass is 137 Da for molecules of the formula 1 , 2 or 3 wherein Ri to R4 are hydrogen atoms.
  • This reporter ion is an important asset of the compounds and methods of the invention because it is easily detected on a mass spectrum, even if the spectrum presents a great number of peaks due to the fragmentation of many biomolecules and biomolecule derivatives possibly present in the sample. DESCRIPTION OF THE DRAWINGS
  • Figure 1 shows the MALDI TOF MS spectrum in positive mode of the commercial carbonylated peptide (PepCO) before and after derivatization with PACa.
  • Figure 2 shows the MALDI TOF MS spectrum in positive mode of the carbonylated A9 peptide after derivatization with PACa.
  • the mass shift of 149.65 Da (1257.25-1107.62) corresponds to the specific interaction between the carbonylated lysine of the A9 peptide and PACa, and a second mass shift of 149.65 Da (1408-1258) corresponds to a second carbonylated lysine derivatized with PACa.
  • Figure 3 shows the MALDI TOF MS spectrum in positive mode of three phospholipids: DMPC (A), PGPC (B) and POVPC (C), before and after reaction with PACa.
  • C, POVPC with and without PACa shows the total derivatization of the carbonylated compound with PACa.
  • Figure 4 shows MALDI MS spectra of (A), pepCO in solution before enrichment on the magnetic silica beads with thiol surface, and (B) after elution from these beads.
  • Figure 5 shows MALDI TOF MS spectra.
  • Spectrum A PACa enrichment of a mix of phospholipids (POVPC, DMPC, PGPC) with an equal ratio placed in the column.
  • Spectrum B the solution collected from the column (POVPC was held on the column).
  • Panel C elution with DTT lOOmM and collecting only the carbonylated phospholipid.
  • Figure 6 shows the UV-visible spectrum from 250 to 500 nm of PACa and a carbonylated protein (human serum albumin), the shift of the maximum of absorbance of PACa from 310 nm when free to 340 nm when fixed to protein carbonyl can be noticed.
  • Figure 7 shows CID MALDI spectrum of the PepCO before (the left spectrum) and after PACa derivatization (the right spectrum), b fragments are the same whereas y fragments shifted by 150.6 Da.
  • the PACa's reporter ion is at 137 Da.
  • Figure 8 shows CID MALDI spectrum of (A), the non modified peptide A9 and (B), the carbonylated lysine derivatized to PACa. Note the presence of the specific signature at 137 Da, the neutral loss of 168 Da (mass of PACa).
  • b and y non modified ions
  • b* and y* PACa derivatized ions
  • ⁇ internal ions
  • ⁇ * internal derivatized ions.
  • Figure 9 shows MS/MS spectrum of POVPC-PACa on the upper spectrum and POVPC on the lower spectrum. Both of them give a fragment at 184.5 Da. The peak at 137 Da signs the presence of PACa.
  • Figure 10 shows MS spectra of carbonylated A9 derivatized with A, PACa, or B, biotin hydrazide.
  • 2 PACa molecules could be fixed on a bicarbonylated peptide whereas only one biotin hydrazide was fixed on the same oxidized peptide.
  • Figure 11 shows MS spectra of the supernatants comprising the oxidized A9 peptide after enrichment on gold beads in presence (GNP+A9ox-PACa) and in absence (GNP + A9ox) of derivatization of the oxidized A9 peptide with PACa.
  • Figure 12 shows MS spectra of the protein digest and A9 oxidized peptide derivatized with PACa (lower spectrum) and of the non retained fraction on GNP (upper spectrum)(a), and MS spectra of the eluted fraction in presence (upper spectrum) and in absence (lower spectrum) of PACa derivatization (b).
  • Figure 13 shows MS spectra of Tau protein (lower spectrum) and of oxidized Tau protein (upper spectrum).
  • Figure 14 shows MS spectra of the_oxidized Tau eluted from the GNP without PACa derivatization (a), the non fixed fraction of oxidized tau-PACa on GNP (b) and the eluted fraction from GNP with oxidized Tau-PACa (c).
  • Figure 15 shows the chemical formulation of PACa with its specific functions (left side), and UV spectrum of PACa with the maximum of absorption at 310 nm (right side).
  • Figure 16 shows ms/ms spectrum of PACa acquired in positive mode by infusion in Q-Tof mass spectrometer.
  • Figure 17 shows the mass spectrum (a) and the MS/MS spectrum (b) of the second synthesized molecule.
  • compounds of the invention have general formula 1.
  • the compounds of the invention are of formulae 1-3 in which Ri, R 2 , R 3 , and R4, identical or different, are hydrogen atoms or (Cl-C8)alkyl groups, more specifically hydrogen atoms.
  • the compounds of the invention are of formulae 4-6 in which Ri, R 2 , R 3 , and R4, identical or different, are hydrogen atoms or (Cl-C8)alkyl groups, more specifically hydrogen atoms.
  • the compound of the invention is of formula 1 in which R l s R 2 , R 3 , and R4 are hydrogen atoms or (Cl-C8)alkyl groups (in particular (Cl-C4)alkyl groups).
  • the compound of the invention is of formula 1 in which Ri, R 2 , R 3 , and R4 are hydrogen atoms (i.e., 4-sulfanylbenzohydrazide).
  • the invention relates to a method for the derivatization, enrichment, identification and/or quantification of carbonyl groups of biomolecules by using a compound as defined above.
  • One method of the invention comprises the following steps: (i) derivatization of biomolecules by the compound of the invention, (ii) identification of specifically carbonylated biomolecules, (iii) optional quantification of carbonylated species and/or of the rate of carbonylation.
  • Derivatization of biomolecules is carried out to form the stable hydrazone.
  • Said step can be represented as follows.
  • bio molecules derivatization by the compound of the invention can be carried out by contacting at least one bio molecule or a sample comprising a mixture of molecules that include biomolecules of interest with at least one compound of the invention.
  • step (i) is performed by contacting a sample comprising a mixture of molecules that include biomolecules of interest with a compound of the invention mounted on a solid support.
  • the compounds of the invention may be mounted on supports, such as thio-silica magnetic beads, gold nanoparticles, or a sepharose column, more specifically via bonds, in particular disulphide bonds, through the thiol groups on the compounds of the invention.
  • the support may be any solid support susceptible to be reacted with the thiol function of the compounds of the invention, for instance any support susceptible to bear thiol groups that can be reacted with the thiol function of the compounds of the invention. Fixation of the compound of the invention on the solid support may also be performed after derivatization of the biomolecule of interest with the compound of the invention.
  • the use of compounds of the invention on a solid support in the method of the invention affords an enrichment of the derivatized biomolecules, thus easing their subsequent analysis.
  • the solid support may be eluted and/or washed with any appropriate solvent or reagent in order to recover a sample comprising a high proportion of biomolecules derivatized with compounds of the invention.
  • Samples containing the biomolecules of interest can be obtained from any source including, but not limited to, any biological or environmental source.
  • the sample may be a biological material, such as fermentation fluid, soil, water, food, pharmaceutical, organ culture, tissue culture, cell culture; any plant tissue or extract including root, stem, leaf, or seed, exhaled breath, whole blood, blood plasma, urine, semen, saliva, lymph fluid, meningal fluid, amniotic fluid, glandular fluid, sputum, feces, sweat, mucous, cerebrospinal fluid, and experimentally separated fractions of all of the preceding solutions or mixtures containing homogenized solid material, such as feces, organs, tissues, and biopsy samples.
  • a biological material such as fermentation fluid, soil, water, food, pharmaceutical, organ culture, tissue culture, cell culture; any plant tissue or extract including root, stem, leaf, or seed, exhaled breath, whole blood, blood plasma, urine, semen, saliva, lymph fluid, meningal fluid, amniotic fluid, glandular fluid
  • Step (ii) may be performed by any characterization technique known in the art.
  • mass spectrometry techniques are used in step (ii).
  • MALDI techniques may be used, in particular MALDI-TOF.
  • mass spectrometry preferably MALDI, in particular MALDI-TOF, is the only characterization technique used in step (ii).
  • Step (ii) is eased by the chemical nature of compounds of the invention that triggers the presence of a reporter ion easily identifiable on mass spectra, as detailed above.
  • MS and MS/MS mass spectrometric techniques are subsequently used to characterize the carbonylated bio molecule fixed with the compounds of the invention.
  • the method of the invention comprises after step (ii) an additional step of using the results of step (ii) to identify biomolecules susceptible to carbonylation by using known methods.
  • the nature or the sequence of the biomolecules susceptible to oxidation may be determined (e.g., by mass spectrometry and other non-mass spectrometry based sequencing techniques).
  • the sequence may subsequently be searched on a sequence database to identify the protein.
  • the sample comprising proteins can be subjected to enzymatic digestion or chemical cleavage, such as with trypsin, resulting in peptides.
  • step (ii) leads to identification of the peptides, when the additional step described in the above paragraph leads to identification of the proteins.
  • the method of the invention allows determination of the number of carbonylated biomolecules out of the total number of biomolecules present in the sample.
  • the method of the invention may be used to determine the number of carbonylated aminoacids per biomolecule of the sample (rate of carbonylation).
  • the aminoacids susceptible to carbonylation are lysine (K), arginine (R), proline (P) and threonine (T).
  • quantification is carried out using UV/Vis spectroscopy and more specifically via the calculation of the Beer Lambert Coefficient of the compound of the invention.
  • the method further comprises the step of (iv) identifying a disease, disorder, or condition associated with the identified biomolecule susceptible to oxidation as determined from step (ii). Identifying a disease, disorder, or condition includes forecasting, detecting, diagnosing or monitoring a disease, disorder, or condition.
  • patient describes an animal, including mammals, for which biomarkers of oxidative stress can be identified using compounds and methods of the present invention.
  • Mammalian species that can benefit from the disclosed methods of the invention include, and are not limited to, humans, apes, chimpanzees, orangutans, monkeys; and domesticated animals such as mice, rats, dogs, cats, guinea pigs, and hamsters.
  • the method may further comprise the step of (iv) identifying a plant pathological situation associated with the identified bio molecule susceptible to oxidation as determined from step (ii). Accordingly, the present invention provides compounds and methods for identifying biomolecules susceptible to, and thus biomarkers of, oxidative stress that may be used to forecast plant pathological situations earlier than the actual manifestation of symptoms.
  • This invention offers a simple, unexpensive method for the analysis of oxidative damage to biomolecules in vitro and in vivo.
  • the PACa compounds react specifically with carbonyls on proteins, peptides as well as lipids to form stable hydrazones.
  • the novel compounds share three important features which make them suitable for the analysis of oxidized biomolecules using mass spectrometry.
  • the PACa compounds have application in the research field for diseases such as Alzheimer's disease and cancer, as specified above, but also in the food industry for the testing the carbonyl content of food. They can be a valuable tool for use in proteomic research, since compounds of the invention can be used to characterize in vivo and in vitro carbonyl contents.
  • the present invention provides a composition comprising at least one compound of the invention and a carrier.
  • composition is contained within a kit.
  • PACa compounds can be used in kits in the form of a pre-loaded solid support, such as a column or beads, preferably nanobeads, developed for the enrichment of biological molecules having low carbonyl content.
  • a “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., benzyl alcohol), antioxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween 80, Polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), water, bulking substance (e.g., lactose, mannitol), excipient, or any auxiliary agent.
  • preservative e.g., benzyl alcohol
  • antioxidant e.g., ascorbic acid, sodium metabisulfite
  • solubilizer e.g., Tween 80, Polysorbate 80
  • emulsifier e.g., Tris HC1, acetate, phosphate
  • water e.g., Tris HC1, acetate, phosphate
  • bulking substance e.g., lactose
  • the present invention provides an article of manufacture where the functionality of a method of the invention is embedded on a computer-readable medium, such as, but not limited to, a floppy disk, a hard disk, an optical disk, a magnetic tape, a PROM, an EPROM, CD-ROM, DVD-ROM, or resident in computer or processor memory.
  • a computer-readable medium such as, but not limited to, a floppy disk, a hard disk, an optical disk, a magnetic tape, a PROM, an EPROM, CD-ROM, DVD-ROM, or resident in computer or processor memory.
  • the functionality of the method can be embedded on the computer-readable medium in any number of computer readable instructions, or languages such as, for example: FORTRAN, PASCAL, C, C++, BASIC and, assembly language.
  • the computer-readable instructions can, for example, be written in a, script, macro, or functionally embedded in commercially available software, (e.g. EXCEL or VISUAL BASIC).
  • a carbonylated commercial peptide [AcN-methyl-YVAD-aldehyde] called PepCO was purchased from Bachem (GMBH, Ge).
  • Another peptide [NH 2 NKPPKKGPANG-OH] called A9 was synthesized.
  • Sodium hypochlorite purchased from Across Organics
  • Bovine Serum Albumin (BSA) purchased from Sigma Aldrich.
  • FeCl 3 , DNPH, HCL, TCA, thiopropylsepharose 6B purchased from GE Healthcare (Sweden).
  • Gold nanoparticles can be purchased from Sigma Aldrich.
  • the compound noted PACa is 4- sulfanylbenzohydrazide, ie the compound of formula (1) with Ri to P being hydrogen atoms.
  • the compound of the invention was synthesized in the following manner:
  • Peptides A9 peptide was oxidized using NaOCl 0.5mM for 20 minutes at 37°C which induced carbonyls.
  • the carbonylated commercial peptide and the oxidized A9 peptide were derivatized with the compound of the invention or conventional biotin hydrazide (10 mM) in HC1 2N for 60 minutes at room temperature, and were then purified and concentrated using a CI 8 stage tips column.
  • Phospholipids - DMPC, PGPC and POOVPC - were dissolved in methanol at 2.5 ⁇ g/ ⁇ l (4.2mM) and treated with the compound of the invention (10 mM in methanol) volume ratio 1 : 1 for 1 hour at room temperature. Samples were then spotted on the MALDI TOF plate 0.5 ⁇ (1 nmol) with 0.5 ⁇ of the DHB matrix (40 mg/ml in 97% ethanol).
  • the sensitivity of the derivatization was estimated by successive dilutions of the POVPC (0.25 ⁇ g/ ⁇ l, 0.025 ⁇ g/ ⁇ l, 0.005 ⁇ g/ ⁇ l and 0.0025 ⁇ g/ ⁇ l) before the reaction of the phospholipid with the compound of the invention (10 mM in methanol) volume ratio 1 : 1 for one hour at room temperature. Samples were spotted on a MALDI TOF plate corresponding respectively to 2.1 nmol, 0.1 nmol, 0.01 nmol, 2 pmol, and 1 pmol on the target.
  • Oxidized Tau protein was derivatized with PACa (10 mM in water) at a volume ratio of 1 : 1, for 1 hour, at room temperature.
  • the compound of the invention (20mM in PBS) was added into the column and the column was left for one hour at room temperature to incubate before being washed with three volumes of the buffer prior to the addition of the carbonylated biomolecule. The biomolecule was then added and allowed to incubate for one hour. A washing step with buffer was subsequently carried out before elution with DTT lOOmM.
  • a solution of Oxidized A9 peptide derivatized with PACa as described above was added togold nanoparticules (GNP), prealably washed once with water.
  • GNP gold nanoparticules
  • the reaction mixture was incubated during 1 hour at room temperature, then centrifugated to recover the GNP in the centrifugation pellet.
  • the recovered GNP were redispersed in water by sonication once for washing, and washed GNP were obtained by centrifugation.
  • the supernatants comprising the oxidized peptide were analyzed by MALDI analysis, by putting ⁇ ⁇ of the eluted samples with 1 ⁇ of a-cyano-4-hydroxycinnamic acid (CHCA) matrix.
  • CHCA a-cyano-4-hydroxycinnamic acid
  • a solution of oxidized Tau protein derivatized with PACa as described above was contacted with gold nanoparticules (GNP), prealably washed once with water, for 1 hour at 25°C under agitation.
  • GNP gold nanoparticules
  • the reaction mixture was centrifugated to recover the GNP in the centrifugation pellet.
  • the recovered GNP were redispersed in water by sonication once for washing, and washed GNP were obtained by centrifugation.
  • Control experiment was performed in parallel with oxidized Tau protein and GNP, without derivatization of the protein with PACa.
  • the supernatants comprising the oxidized Tau protein were analyzed by MALDI analysis. The obtained spectra are presented on figure 14.
  • the whole process from steps 1 to 4 as described above can be monitored by mass spectrometry techniques, as is demonstrated in this section.
  • the PACa product obtained by chemical synthesis was 4-sulfanylbenzohydrazide, (C 7 H 8 O 1 N 2 S 1 with a molecular mass 168.2174 g mol "1 ) and is a white powder which is soluble in water up to 13 mM, in methanol up to 49 mM, and shows higher solubility in DMSO.
  • the "trapping" step of the invention involves the reaction of carbonyl groups with the PACa molecules to form marked carbonyl groups that are later counted in the "quantification” step. This reaction is known as derivatization and is here described with reference to figures 1-3.
  • Figure 2 relates to the derivatization with peptide A9.
  • NaOCl carbonylation of the A9 peptide on one lysine induces a mass loss of 1.03 Da
  • the net mass shift with PACa derivatization is then 149.57 Da.
  • Figure 2 shows the MALDI TOF MS spectrum in positive mode of the carbonylated A9 peptide.
  • the PACa fixed to the A9 carbonylated peptide gave species at 1257.25 Da and 1406.89 Da corresponding to one and two sites of carbonylated lysines derivatized with PACa.
  • Figure 3 relates to the derivatization with phospholipids.
  • Three phospholipids: DMPC (A), PGPC (B) and POVPC (C) were analyzed by MALDI-Tof MS before and after reaction with PACa.
  • POVPC is a carbonylated phospholipid and therefore should be the only one to react with PACa.
  • Figure 3 clearly showed that the derivatization worked only with the carbonylated compound POVPC and not with the others.
  • Figure 4 demonstrates PACa's enrichment of the PepCO.
  • a support of thio-silica magnetic beads was used to test the enrichment capacity of PACa to the PepCO. Firstly, the beads were contacted with PACa to form disulfide bonds. PepCO was then mixed with the beads for 90 minutes. Beads were subsequently washed, then eluted with DTT (see Fig. 4, B), the expected peaks were present at masses: 679.55 Da, 695.55 and 717.55; corresponding to PACa-PepCO+Na + , PACa-PepCO+K + and PACa-PepCO+Na + +K + .
  • Figure 5 shows PACa's enrichment of phospholipids.
  • Figure 5 A shows a MALDI spectrum in the positive mode for a mix of three phospholipids with an equal ratio in methanol and DHB matrix: a carbonylated lipid POVPC (594.3 and 616.3 Da for MH + and M+Na + ), as well as the other lipids PGPC (610.4, 632.3 and 654.3 Da for MH + , M+Na + and M+2Na + ) and DMPC (678.5, 700.4 and 716.4 Da for MH + , M+Na + and M+K + ).
  • This mixture was passed through a PACa-sepharose column prepared in house to enrich and isolate specifically the POVPC.
  • Figure 5 B shows the remainder of species from the mixture after passing through the PACa- column.
  • the quantification of protein carbonyls was carried out using UV/Vis spectroscopy.
  • the results for quantification of carbonyl contents were similar for both DNPH and PACa (see Fig.6).
  • Figure 7 demonstrates the MALDl TOF TOF characterization of the PepCO fixed to PACa. It is the CID MALDl spectrum of the PepCO before and after reaction with PACa. Because of the location of the carbonyl at the C-terminus end, solely 'y' fragments were shifted by 150.6 Da. One can observe a specific daughter ion signature for PACa at 137 Da.
  • FIG. 8 shows the CID MALDl spectrum of the A9 peptide on the upper spectrum and the derivatized carbonylated lysine with PACa on the lower spectrum.
  • Figure 10 compares the reactivity of PACa and biotin hydrazide to carbonyl using the same oxidized A9 peptide.
  • Figure 11 presents the MALDl analysis of the supernatants comprising the oxidized A9 peptide after enrichment on gold beads in presence (GNP+A9ox-PACa) and in absence (GNP + A9ox) of derivatization of the oxidized A9 peptide with PACa.
  • the control spectrum in absence of derivatization with PACa comprises only background noise.
  • the spectrum in presence of PACa derivatization presents peaks at m/z 1214.20 and 1257.21 that are characteristic of carbonylated A9 peptide. Fixation of PACa on gold beads thus allowed specific enrichment of oxidized A9 peptide.
  • the presence of the m/z 1257.27 peak in the lower spectrum of the MS spectra of figure 12 a) and its absence in the upper spectrum shows that the oxidized A9 peptide derivatized with PACa is preferentially retained on the GNP when compared to the rest of the (protein digest + oxidized A9 peptide) mixture.
  • the presence of the m/z 1214.2 and 1257.27 peaks in the upper spectrum of the MS spectra of figure 12 b) shows an efficient enrichment of the carbonylated A9 peptide derivatized with PACa was obtained on the GNP.
  • the m/z 1046.5 and 1672.9 peaks correspond to the signature of a peptide mix used for calibration.
  • the PACa scaffold is composed of three main components, (see Fig. 15):
  • a hydrazide function that reacts specifically with carbonyls to form a stable hydrazone at acidic pH.
  • thiol function for the enrichment of carbonyls by disulfide linkage on thiol activated chromatographic support.
  • PACa compounds are able to specifically react with more than one modified amino acid on a peptide sequence and are far less limited than conventional derivatizing agents with respect to steric hindrance.
  • PACa is 4-sulfanylhydrazide
  • studies on the carbonylated A9 peptide sample at the same concentration for each derivatizing agent; PACa and biotin hydrazide demonstrated that two molecules of PACa could be fixed onto the peptide whereas only one molecule of biotin hydrazide could be fixed (Fig. 10).
  • PACa bound to a carbonyl gives rise to a specific MS/MS signature at 137 Da, which is highly practical because this mass neither matches any charged amino acid mass fragment, nor any immonium ion fragment.
  • This is of particular interest for the analysis of MS/MS modified amino acids. Easy characterization and quantification of modified substances is possible with UV testing due to the specific absorbance at 310 nm in water and buffers.
  • the PACa compounds have excellent solubility characteristics which allow the use of organic solvents such as methanol and DMSO, as well as with pure water or biological buffers.
  • the solubility is highly compatible with in vivo cellular analyses.
  • the solubility is excellent in comparison with other previously engineered compounds, for example, biotin hydrazide.
  • the PACa compounds show similar specificity to the conventional derivatizing agent DNPH, the latter does not allow enrichment of the carbonylated biomolecular species. Elution following biotin hydrazide coupling and enrichment via Streptavidin beads was attempted and was unsuccessful due the strong affinity between the two partners that prevented efficient release. By contrast, the enrichment with PACa compounds is easy, and the elution from thio binding materials occurs via simple pH steps.

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Abstract

La présente invention concerne des composés et des procédés pour la transformation en dérivés, l'identification et/ou la quantification de biomolécules carbonylées à l'aide des composés de l'invention. L'invention concerne des composés et un procédé d'utilisation de tels composés pour l'analyse d'espèces biomoléculaires oxydées (telles que les peptides, les protéines et les lipides). Ce procédé permet, de façon avantageuse, une transformation en dérivés, une quantification et un enrichissement simultanés d'échantillons carbonylés et se prête totalement à l'analyse par spectrométrie de masse.
PCT/EP2012/058844 2011-05-11 2012-05-11 Composés et procédés pour l'identification et/ou la quantification de biomolécules carbonylées Ceased WO2012152943A1 (fr)

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GB2523114A (en) * 2014-02-12 2015-08-19 Kratos Analytical Ltd Oxidized lipid detection
CN115932131A (zh) * 2023-01-16 2023-04-07 南京农业大学 一种肌肉组织中羰基化蛋白质的富集和鉴定方法
CN116554076A (zh) * 2023-05-10 2023-08-08 复旦大学 酰肼类化合物作为衍生化试剂的应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2523114A (en) * 2014-02-12 2015-08-19 Kratos Analytical Ltd Oxidized lipid detection
WO2015121627A1 (fr) * 2014-02-12 2015-08-20 Kratos Analytical Limited Détection de lipides oxydés
US10948502B2 (en) 2014-02-12 2021-03-16 Kratos Analytical Limited Oxidized lipid detection
CN115932131A (zh) * 2023-01-16 2023-04-07 南京农业大学 一种肌肉组织中羰基化蛋白质的富集和鉴定方法
CN116554076A (zh) * 2023-05-10 2023-08-08 复旦大学 酰肼类化合物作为衍生化试剂的应用

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