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WO2007132164A2 - Analyse de protéines - Google Patents

Analyse de protéines Download PDF

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Publication number
WO2007132164A2
WO2007132164A2 PCT/GB2007/001621 GB2007001621W WO2007132164A2 WO 2007132164 A2 WO2007132164 A2 WO 2007132164A2 GB 2007001621 W GB2007001621 W GB 2007001621W WO 2007132164 A2 WO2007132164 A2 WO 2007132164A2
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WO
WIPO (PCT)
Prior art keywords
peptide
marker
seq
marker peptide
nos
Prior art date
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Ceased
Application number
PCT/GB2007/001621
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English (en)
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WO2007132164A3 (fr
Inventor
Peter Bramley
Paul Fraser
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Royal Holloway and Bedford New College
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Royal Holloway and Bedford New College
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Filing date
Publication date
Priority claimed from GB0608602A external-priority patent/GB0608602D0/en
Priority claimed from GB0608604A external-priority patent/GB0608604D0/en
Application filed by Royal Holloway and Bedford New College filed Critical Royal Holloway and Bedford New College
Publication of WO2007132164A2 publication Critical patent/WO2007132164A2/fr
Publication of WO2007132164A3 publication Critical patent/WO2007132164A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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

Definitions

  • the present invention generally relates to the detection of genetically modified plant-derived material in products. More specifically, the invention relates to the identification of transgenic proteins present in plant-derived materials.
  • the present invention relates to a method for detecting the presence of the transgenic protein 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in a sample of a plant-derived material, a marker peptide derived from EPSPS and a method for isolating a marker peptide from EPSPS, use of the peptide markers in the detection of EPSPS and a kit suitable for the detection, identification and quantification of EPSPS in a plant-derived material.
  • EPSPS transgenic protein 5-enolpyruvylshikimate-3-phosphate synthase
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase ⁇
  • S3P shikimate-3- phosphate
  • phosphoenolpyruvate to 5-enolpyruvylshikimate-3-phos ⁇ hate
  • Quantitative competitive PCR as well as real-time quantitative PCR have been employed broadly for the detection of transgenic DNA. Whilst PCR methodologies are very sensitive, their reliability depends on the integrity of the DNA, which can be degraded by heat, nuclease activity and low pH.
  • Protein immunoassays are utilized particularly in the food industry because of their ease-of-use, high sensitivity and capability to high-throughput.
  • a suitable antibody is not always easily available, especially for new target proteins, and the effect of food processing, heat, pH, etc., to the validity of the assay needs to be investigated in each case. Whilst these external parameters might also pose a problem for other types of protein analysis, the immunoassay methods additionally might suffer from non-specific binding and cross contamination.
  • a method for determining the presence of genetically-modified plant-derived material in a product comprising: providing a protein extract derived from the product; enriching the protein extract; - digesting the protein extract using an enzyme; and detecting the presence or absence of at least one marker peptide resulting from the enzymatic digestion of a transgenic protein thereby determining whether the genetically modified plant derived material is present in the product.
  • the method according to the invention provides a practical, low-cost, accurate and robust proteomic approach to the detection of genetically-modified plant-derived material in a product. Furthermore, the method can be readily automated to provide an analytical approach for routine surveillance in the laboratory for the identification and detection of a transgenic protein (GM component) at levels established by regulatory authorities (i.e. ⁇ 0.9%). This is particularly the case when the product is a processed food product.
  • the method can also be used for multiplex studies where a plurality of marker peptides or transgenic proteins can be analysed in the same analytical sample.
  • the invention also provides: a marker peptide for use in determining the presence of genetically- modified plant-derived material in a product, the marker peptide having a sequence selected from SEQ ID Nos.
  • compositions for use in determining the presence of genetically modified plant material in a product comprising two or more marker peptides of the invention; a marker peptide: ⁇ olymeric adsorbent complex, wherein the marker peptide has a sequence selected from SEQ ID NOs. 1 to 18 and oxidised variants thereof; a method for isolating a marker peptide or composition of the invention comprising: providing a protein extract derived from a plant-derived material comprising 5-enolpyruvylshikimate-3-phosphate synthase (CP4
  • EPSPS EPSPS
  • enriching the protein extract and digesting the protein extract with an enzyme
  • use of a marker peptide, composition or complex of the invention for detecting the presence of genetically modified plant-derived material in a product and a kit for detecting the presence of a genetically modified plant- derived material in a product, the kit comprising: a digestive enzyme; an enzyme solubilisation reagent; an enzyme reaction buffer; and at least one reference peptide marker or a computer-readable algorithm capable of elucidating a mass spectrometric signal of at least one reference peptide marker.
  • FIG. 1 shows a reaction scheme illustrating the inhibition of EPSP synthase by glyphosate.
  • EPSPS 5-Enolpyruvylshikimate-3-phosphate synthase
  • S3P shikimate-3 -phosphate
  • PEP phosphoenolpyruvate
  • ESP 5-enolpyruvylshikimate-3-phosphate
  • This step is inhibited by glyphosate, which is the active ingredient in Roundup ReadyTM herbicides.
  • Figure 2 shows the fractionation of GM soya proteins.
  • Gel filtration fractions (A) absorbance measurements at 280 run (grey line) and 595 nm (black line) (B) SDS-PAGE.
  • Gel filtration fraction 12 was collected within the column void. Potential CP4 EPSPS SDS-PAGE bands at about 47 kDa were present in gel filtration fractions 27 and 29 (B) and anion exchange fractions 75, 80, 82 and 90 (D).
  • Figure 3 shows MALDI-TOF mass spectra of tryptic peptide maps of 47 kDa SDS-PAGE bands from 100% by weight GM soya.
  • A Crude protein extract,
  • B gel filtration fraction 27 and
  • C anion exchange fraction 82.
  • the detected CP4 EPSPS tryptic peptides in (B) and (C) are labeled by mass and residue number.
  • Figure 4 shows NanoLC-nanoESI-QTOF mass spectra of in-solution digested anion exchange fraction 92 from 50 % by weight GM soybean seeds.
  • CP4 EPSPS biomarkers (A) [M+2H] 2+ at m/z 558.297 ([M+H] + at m/z 1115.605) and (B) [M+2H] 2+ at m/z 779.933 were observed. Their characteristic MS/MS spectra were used as fingerprints for identification of CP 4 EPSPS (C, D).
  • Figure 5 shows TOF MS ion extracted chromatograms (XIC) of combined anion exchange fractions 82 and 86 from non-GM and 0.9 % by weight GM soybean seeds.
  • XIC TOF MS ion extracted chromatograms
  • Figure 6 shows SDS-PAGE of various anion exchange fractions from 100% by weight GM maize. Potential CP4 EPSPS SDS-PAGE bands at about 47 kDa were present in fractions 79 and 81.
  • Figure 7 shows a MALDI-TOF mass spectrum of a 47 kDa SDS-PAGE band from anion exchange fraction 81 from 100% by weight GM maize.
  • the detected CP4 EPSPS tryptic peptides are labeled by mass and residue number.
  • Figure 8 shows a graph of the ratio of the intensity of the molecular ions
  • Figure 9 shows a comparison of experimental and theoretical ratios for EPSPS in GM soya using AQUATM labelling.
  • the invention provides a method for determining the presence of genetically-modified plant-derived material in a product, the method comprising: providing a protein extract derived from the product; enriching the protein extract; digesting the protein extract using an enzyme; and - detecting the presence or absence of at least one marker peptide resulting from the enzymatic digestion of a transgenic protein thereby determining whether the genetically modified plant derived material is present in the product.
  • Advantages according to the method of the present invention are that surprisingly the detection of GM materials present in a sample at amounts as low as 0.5% by weight of the sample are possible.
  • the peptide with the sequence of SEQ ID No. 13 was detected in accordance with method of the present invention in 0.5% by weight genetically-modified soya.
  • 0.9% by weight is currently the recognised lower quantitative limit (and the European Union regulatory limit for foodstuffs) for the detection of GM materials in material available from methods known in the art, hence this is a significant improvement over known methods.
  • the genetically modified plant derived material is detected by determining the presence or absence of one or more peptide markers derived from one or more transgenic protein.
  • the present invention also provides for the analysis of a plant-derived material to determine the presence of a transgenic protein, comprising the steps of: providing a protein extract derived from a plant-derived material potentially comprising a transgenic protein; enzymatic digestion of the protein extract suitable to produce a marker peptide; and detection of the presence of the marker peptide to determine the presence of a transgenic protein using at least one reference marker peptide.
  • the genetically modified plant-derived material may be from any plant.
  • the plant is a crop plant such as, for example, soyabean, maize, corn, barley or wheat.
  • the plant-derived material is from soyabean or maize.
  • the product is, in one preferred embodiment, a food matrix.
  • the product employed according to the method of the invention may be maize or soyabean seed, typically genetically modified maize or soybean seed.
  • the food matrix typically contains GM ingredients.
  • the food matrix may be a processed foodstuff such as bread, cake, biscuit, confectionery, a processed meat product (eg, a sausage), packaged dehydrated noodles and the like. It may also be a livestock feedstuff to be used in agriculture. Furthermore, the matrix may be derived from farm animal waste (eg, cow faeces).
  • the wide variety of plant-derived materials that the method of the present invention can be applied to demonstrates its versatility and usefulness in analysing all manner of materials ranging from protein isolates to raw ingredients to complex food matrices such as processed foods which are on the market.
  • the method of the invention may also be applied to environmental surveillance studies, for example, as part of the labroratory analysis of farm animal waste.
  • the product may comprise up to 100% plant-derived material, such as from
  • the product may contain plant- derived material from one, two or more different types of plant.
  • the method may be used to detect transgenic proteins from each type of plant present in the product.
  • the method of the invention includes the step of enrichment of the protein extract. Preferably, this takes place prior to enzymatic digestion.
  • the enrichment of the transgenic protein from the protein extract may comprise the steps of gel filtration chromatography, anion exchange chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
  • gel filtration and SDS-PAGE are used to enrich the protein extract.
  • anion exchange chromatography is also used prior to SDS-PAGE.
  • Enrichment may alternatively or additionally take place after enzymatic digestion.
  • the peptides present after digestion may, for example, be enriched using reverse phase nano liquid chromatography.
  • Enrichment is particularly important where the presence of the transgenic protein of interest is suppressed by the presence of other proteins which have a substantially higher abundance in the sample. This is of particular relevance for transgenic proteins present in GM soya, or other seeds or pulses, due to the abundant presence of major storage proteins.
  • the above enrichment techniques for biomolecules (including proteins) are individually well known to the person skilled in the art.
  • the marker peptide is a transgenic marker peptide resulting from the enzymatic digestion of the transgenic protein to be detected.
  • enzymatic digestion employed according to the method of the invention utilises an enzyme suitable for digesting the protein into an analysable peptide fragment.
  • Suitable enzymes will be known to the person skilled in the art.
  • trypsin, endoproteinase AspN and/or endoproteinase GIu-C are employed.
  • trypsin is used for enzymatic digestion.
  • the transgenic protein being detected is 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), preferably the transgenic protein CP4 EPSPS derived from Agrobacterium tumefaceins CP4.
  • EBP 5-enolpyruvylshikimate-3-phosphate synthase
  • CP4 EPSPS derived from Agrobacterium tumefaceins CP4.
  • the marker peptide or peptides of the present invention are derived from 5-enol ⁇ yruvylshikimate-3-phosphate synthase (EPSPS PS).
  • EPSPS PS 5-enol ⁇ yruvylshikimate-3-phosphate synthase
  • the method of the invention may employ a marker peptide according to the invention. Therefore, in one embodiment, the method of the present invention employs at least one marker peptide having a sequence selected from SEQ ID NOs. 1 to 18 and including oxidised variants thereof.
  • the marker peptide may have a sequence selected from SEQ ID NOs 2, 3, 8, 13 and oxidised variants thereof.
  • the detection of the marker peptide is by mass spectrometric analysis.
  • Mass spectrometric analytical methods employed according to the method of the invention include matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and/or nanoelectrospray ionisation quadrupole time-of-flight (nanoESI-QTOF) mass spectrometry and/or nano liquid chromatography nanoelectrospray ionisation quadrupole time-of-flight tandem mass spectrometry (nanoLC-nanoESI-QTOF MS/MS) and/or ion trap mass spectrometry.
  • MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight
  • nanoESI-QTOF nanoelectrospray ionisation quadrupole time-of-flight
  • nanoLC-nanoESI-QTOF MS/MS nano liquid chromatography nanoelectrospray ionisation quadrupole time-of-flight tandem mass spectrome
  • the mass spectrometric techniques employed in the present invention available for the analysis of proteins in materials require only a minute amount of sample, may be easily automated, and can provide highly detailed information in relation to samples of proteins including high mass accuracy.
  • MALDI-TOF MS is a soft ionization technique used in mass spectrometry, which ionizes biomolecules (eg, biopolymers like proteins, peptides and sugars) which tend to lose their structural integrity when ionized by conventional ionization methods.
  • biomolecules eg, biopolymers like proteins, peptides and sugars
  • Electrospray ionization (ESI) mass spectrometry is a technique useful in producing ions from large biomolecules because it overcomes the propensity of these molecules to fragment when ionized.
  • ESI mass spectrometry quasimolecular ions are observed that are ionized by the addition of a proton to give [M+H], or other cation (eg, a sodium ion) to give [M+cation], or the removal of a proton [M-H].
  • multiply charged ions such as [M+2H] may be frequently observed.
  • Quadrapolar ion trap mass spectrometric techniques may also be employed for mass spectrometric analysis according to the methods of the present invention.
  • Mass spectrometry can identify large and small molecules and assists in determining their molecular structure, providing a sensitive and versatile means for analysing complex biological mixtures including protein and peptide mixtures.
  • Ions can be created by electron impact (EI), electrospray (ESI), or matrix-assisted laser desorption (MALDI) ionization.
  • Quantitative mass spectrometric analysis may be used in a method of the invention to determine the amount of marker peptide present. This is preferably done by using a known amount of a reference marker peptide in a first sample, providing a second sample potentially comprising a quantity of the marker peptide, subjecting the first and second samples to mass spectrometric analysis to produce a mass spectrometric signal, and making a quantitative comparative measurement of the intensity of the mass spectrometric signal produced by the first and second samples in order to determine the quantity of marker peptide present in the second sample and hence transgenic protein present in a plant-derived material.
  • amine-specific labeling reagents eg, iTRAQTM reagents (Applied Biosystems)
  • stable isotope labeled peptides eg, Protein-AQUATM
  • reagents and the methodologies for employing them as means for the quantitative protein analysis are known to the person skilled in the art.
  • the reference marker peptide is labeled.
  • Amine-specif ⁇ c labeling reagents may be employed for relative protein quantification using mass spectrometry.
  • reagents are a primary amine specific label that covalently binds to lysine side chains and the N-terminal group of a peptide.
  • the labelled peptides are used as an internal standard when quantitatively analysing a sample containing a protein of interest.
  • the sample to be analysed may contain the labelled sample control and can be subjected to the purification, enzymatic digestion and mass spectrometric steps discussed hereinbefore.
  • the quantity of the protein of interest present in the sample being tested may then be evaluated by a comparison of the ratio of the intensities (% abundance) of the molecular ion peak for the non-labelled peptide present in the sample being tested (eg, LAGGED V ADLR derived from EPSPS) and the amine- specific labelled peptide standard which was added in a known amount to the sample prior to analysis.
  • the non-labelled peptide present in the sample being tested eg, LAGGED V ADLR derived from EPSPS
  • Stable isotope labeled ( 13 C and 15 N) peptides may be used as an internal standard with a single labelled amino acid per peptide.
  • a specific example of a labelled peptide used in accordance with the present invention is
  • a labelled peptide such as L* AGGED VADLR for the detection of EPSPS
  • the sample may then be subjected to the purification, enzymatic digestion and mass spectrometric steps discussed hereinbefore.
  • the lebelled peptide may alternatively be added immediately prior to mass spectrometric analysis.
  • the amount of the protein of interest present in the sample being tested could then be quantified by a comparison of the ratio of the intensities of the molecular ion peak for the non-labelled peptide present in the sample being tested (e.g.
  • the mass spectrometric analysis step of the method of the invention comprises a step of elucidating a mass spectrometric signal from that of a known transgenic protein by means of computer analysis.
  • This may comprise a step of providing a computer-readable algorithm able to elucidate a mass spectrometric signal resulting from a sample known to contain the transgenic protein (e.g. CP4 EPSPS).
  • CP4 EPSPS transgenic protein
  • the signal from the mass spectrometer is transferred to a computer which contains a program which converts the signal into a computer-readable form.
  • the software may include an algorithm which can identify whether a particular peak in the mass spectrum of the sample corresponds to a peptide marker of the invention. In some instances, such analysis may normally be done manually by observation with the eye. However, automated detection of peaks when multiple molecular ions are generated in the mass spectrometer can be a complex task. Hence, the provision of computer-aided analytical means as described can be of great benefit to the user in saving time and reducing the risk of error in the determination of results.
  • the software can also include a code which distinguishes between a typical mass spectrometric signal characteristic of a known peptide marker or mixture of peptide markers present in a sample and a labeled peptide introduced (spiked) into the sample for quantitative analysis purposes.
  • MALDI-TOF mass spectrometry is a preferred form of mass spectrometry, particularly where the transgenic protein is CP4 EPSPS.
  • peptide sequence information is subsequently obtained by nanoLC- nanoESI-QTOF MS/MS.
  • MALDI-TOF mass spectrometry is a preferred method for the identification of a marker peptide with a sequence selected from SEQ ID NOs. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 16 and 17.
  • NanoESI-QTOF is a preferred method for the identification of a marker peptide with a sequence selected from SEQ ID NOs. 1, 2, 3, 4, 5, 7, 8, 9, 11, 13, 15 and 18.
  • NanoLC-nanoESI-QTOF MS/MS is a preferred method for the identification of a marker peptide with a sequence selected from SEQ ID NOs. 2, 3, 6, 8, 10, 12, 13, 15, 16 and 17.
  • detection of the marker peptide according to the method of the invention comprises isolation of the marker peptide by a molecular imprinting technique using a polymeric adsorbent, wherein the method comprises: labelling the isolated marker peptide with a fiuorophoric or chromophoric molecular label; and spectroscopic detection of the presence of the marker peptide.
  • Molecular imprinting is a technique for creating molecular recognition sites in a polymeric material.
  • the molecule of interest may act as a template for assembling polymerisable functional monomelic units (eg, acrylate monomers) in non-covalent or weakly covalent interactions to form a complex with the template.
  • the monomelic units may then be polymerised, eg, by cross-linking the monomelic units, and the marker peptide template extracted to provide a polymeric substrate (polymeric solid-phase extraction (SPE) adsorbent) with a vacant recognition site corresponding to the extracted marker peptide.
  • SPE polymeric solid-phase extraction
  • the polymeric adsorbent can then act as a selective adsorbent for isolating the same marker peptide present in a sample being analysed which contains a mixture of peptides and/or other biomolecules including the marker peptide of interest. This provides an effective means for the selective enrichment of a transgenic protein or marker peptide derived from a transgenic protein present in a sample comprising an abundance of different biomolecules.
  • the identification and selection of suitable polymeric adsorbents for marker peptides according to the invention is preferably undertaken on pre-synthesised libraries of adsorbents or by a computational design approach.
  • the computational design approach uses an algorithm for determining likely interactions of a virtual library of polymeric adsorbents with a marker peptide of interest. Identification of new potential adsorbent can be achieved and synthesis of a polymeric adsorbent according to the general method outline above can subsequently be undertaken.
  • Marker peptides according to SEQ ID NOs 1 to 18 in quantities sufficient for studies enabling identification and selection of suitable polymeric adsorbents may be prepared by protein synthesis routes known in the art. In the methods of the invention, in a sample which is a crude digested protein extract, a marker peptide is able to complex (bind) in the recognition site of the polymeric adsorbent. Non-complementary molecules remain unbound.
  • Separation of a resultant polymeric adsorbent:marker peptide complex from the sample and physical separation (eg, by solvent elution) of the peptide from the adsorbent provides an efficient means for isolating a marker peptide from a sample containing numerous other peptides and/or biomolecules in abundance. Further purification of the marker peptide can be performed by HPLC or other suitable chromatographic technique(s). The isolated marker peptide may then be labelled with a fluorophoric or chromophoric molecular label.
  • Suitable fluorophoric or chromophoric labels include 5-dimethylaminonaphthalene-l -sulphonyl chloride (Dansyl reagent) or Cascade Yellow succinimidyl ester.
  • Other suitable fluorophoric or chromphoric labels are well known to the skilled person as are methodologies for attaching a label to a peptide. Bonding of the molecular label to the marker peptide will typically be at the primary amine site at the N-terminus or the carboxylate site at the C-terminus of the peptide.
  • the marker peptide may be labelled while still in a crude digested protein extract prior to isolation by a molecular imprinting technique.
  • Spectroscopic detection of the marker peptide may be undertaken using techniques such as ultra-violet/visible (UV/Visible) spectroscopy to detect chromophore-labelled species and fluorescence spectroscopy for detecting fluorophore-labelled species.
  • techniques such as ultra-violet/visible (UV/Visible) spectroscopy to detect chromophore-labelled species and fluorescence spectroscopy for detecting fluorophore-labelled species.
  • peptides especially those of less than 10 amino acids
  • chromphores fluorophores or electrophores.
  • their detection by UV absorption between about 205 to about 230 nm often relies on the presence of a peptide binding carbonyl group. If an aromatic side chain is present then detection may be possible at wavelengths of about 250 to about 280 nm.
  • derivatisation of the marker peptides by incorporating a fluorophoric or chromphoric molecular label improves selectivity and sensitivity of the analysis by increasing the ultraviolet/visible wavelength range of absorbance and absorbance signal intensity, thereby avoiding signals resulting from interference by other substances.
  • the reference marker peptide used in the method of the invention also contains a fluorophoric or chromphoric label.
  • the acquisition of mass spectrometric, spectroscopic and chromatographic data may be used to confirm the nature of a marker peptide which has been labelled.
  • spectroscopic detection of the marker peptide detects the amount of marker peptide present using quantitative analysis by UV/Visible spectroscopy or fluorescence spectroscopy.
  • this is by means of acquiring a spectrum of a sample solution containing a labelled marker peptide analyte in an unknown quantity and measuring the absorbance of the solution. The concentration of the labelled marker peptide in the solution can then be determined if the extinction coefficient and path length through the solution are known.
  • the method of analysis according to the invention does not employ an enzyme-linked immunoabsorbent assay or other immunoassay-based analytical procedure for the detection of the marker peptide.
  • the invention provides a marker peptide for use in determining the presence of genetically modified plant-derived material in a product, the marker peptide having a sequence selected from: SSGLSGTVR (SEQ ID NO. 1);
  • VLMPLR (SEQ ID NO. 5);
  • VLNPLREMGVQVK (SEQ ID NO. 6);
  • EMGVQVK SEQ ID NO. 7
  • SEDGDRLPVTLR SEQ ID NO. 8
  • MLQGFGANLTVETDADGVR SEQ ID NO. 12
  • LAGGEDVADLR SEQ ID NO. 13
  • GLGNASGAAVATHLDHR SEQ ID NO. 17
  • IELSDTK SEQ ID NO. 18
  • oxidised variants thereof '
  • a marker peptide of the invention may also be used to detect the presence of a transgenic protein in a plant-derived material.
  • the marker peptides according to the invention are stable and retain their structural intregrity while being subjected to a range of analytical procedures and chemical modifications making them effective candidates for the identification of EPSPS during the laboratory analysis of complex biomolecular matrices, eg, processed foodstuffs.
  • the marker peptide according to the invention is a marker peptide having a sequence selected from SEQ ID NO. 2, 3, 8 and 13.
  • the peptide marker is derived from 5-enolpyruvylshikimate-3 -phosphate synthase (CP4 EPSPS) sourced from Agrobacterium tumefaciens CP4.
  • CP4 EPSPS 5-enolpyruvylshikimate-3 -phosphate synthase
  • an oxidised variant of a marker peptide according to the invention there is provided.
  • the invention also provides a composition comprising two or more marker peptides of the invention.
  • Preferred marker peptide mixtures which are particularly useful in mass spectrometric analyses, are mixtures comprising at least one of:
  • An additional useful peptide may result from a conservative modification to the amino acid sequences of a marker peptide according to the invention.
  • a conservative modification refers to a change in an amino acid residue which does not alter the polarity or charge of the residue. Examples of conservative changes are well known to those skilled in the art, and include, for example, substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine.
  • conservative amino acid changes are considered to be identical amino acids. Therefore, a conservative amino acid change in amino acid sequence will not affect the percentage amino acid identity between the two sequences. A conservative modification will produce a peptide having functional and chemical characteristics similar to those of the master peptide.
  • a marker peptide according to the invention may be readily applied to a practical and easy-to-use kit for detecting, eg, a transgenic protein in a sample derived from a food matrix such as a food ingredient or a processed foodstuff.
  • the marker peptide according to the invention will comprise a covalently bonded fluorophoric or chromophoric label or be labelled with an isotopically-enriched substituent.
  • fluorophoric labels include dimethylaminonaphthalene-1 -sulphonyl chloride (Dansyl reagent) or Cascade Yellow succinimidyl ester. Isotopic enrichment may be with 2 H, 13 C, 15 N or 18 O isotopes. 15 N enrichment is preferred.
  • a marker peptide with a sequence selected from SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, including oxidised variants thereof, further comprising a covalently bonded fluorophoric or chromophoric molecular label, or isotopically-enriched label.
  • the method of isolating the marker peptide according to the invention will further comprise the step of isolating the marker peptide by a molecular imprinting technique using a polymeric adsorbent. More preferably, this will include forming a marker peptide:polymeric adsorbent complex and separating the marker peptide from the polymeric adsorbent by solvent elution. According to the invention, there is also provided a marker peptide:polymeric adsorbent complex, wherein the marker peptide has a sequence selected from SEQ ID no. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, including oxidised variants thereof.
  • a peptide marker according to the invention or a marker peptide mixture according to the invention as a marker for detecting the presence of a transgenic protein extracted from a plant-derived material
  • a method for isolating the marker peptides or marker peptide mixtures according to the invention comprising the steps of: providing a protein extract derived from a plant-derived material comprising 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS); and enzymatic digestion of the protein extract.
  • CP4 EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • kits for detecting the presence of a transgenic protein extracted from a plant-derived material comprising: a digestive enzyme; an enzyme solubilisation reagent; an enzyme reaction buffer; and a peptide marker as a reference comprising a marker peptide with a sequence selected from SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, including oxidised variants thereof.
  • kits for the mass spectrometric detection and identification of a transgenic protein in a plant-derived material comprising: a digestive enzyme; - an enzyme solubilisation reagent; an enzyme reaction buffer; and a computer-readable algorithm capable of elucidating a mass spectrometric signal of a peptide marker reference with a sequence selected from SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, including oxidised variants, and any combination thereof.
  • the kit according to the invention will include an amine-specific labelling reagent and/or a synthetic peptide and/or a stable isotope labelled peptide for the quantitative analysis of protein present in a sample. Examples of such reagents are the iTRAQTM and Protein-AQUATM reagents.
  • the kit according to the invention may also comprise solvents such as trifluoroacetic acid solution (0.1 to 1.0 %) and acetonitrile.
  • the kit according to the invention may also comprise a matrix material for holding a sample to be analysed.
  • Suitable matrix materials which apply to MALDI- TOF mass spectrometry include ⁇ -cyano-4-hydroxycinnamic acid, 3,5-dimethoxy- 4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid and sinapinic acid.
  • Other suitable matrix material will be known to the skilled person.
  • Suitable enzyme solubilisation reagents include SDS, Triton X- 100, Tween 20, Tween 40, Tween 60 and Tween 80, cholic acid and deoxycholate. Other suitable solubilisation re-agents will be known to the skilled person.
  • enzyme reaction buffers include 100 mM Tris-HCl (pH 8.0),
  • TEAB tetraethylammonium bromide
  • 5OmM ammonium bicarbonate pH 8.0
  • Other suitable buffers will be known to the skilled person.
  • Tris(hydroxymethyl)methylamine Tris-HCl
  • analytical grade acetone and ethanol HPLC-grade acetonitrile
  • ACN HPLC-grade acetonitrile
  • bromophenol blue trifluoroacetic acid
  • Ultrapure protogel and concentrated 10 X Tris/Glycine/SDS (electrophoresis grade) were from National Diagnostics (Hessle, UK).
  • 0.45 ⁇ m Syringe filters were obtained from Fischer Scientific (Loughborough, UK) and glass micro fiber filters from Whatman (Brentford, UK).
  • HiLoad 26/60 SuperdexTM 75 gel filtration column and Q Sepharose resin were from Amersham Biosciences (Little Chalfont, UK).
  • Modified trypsin (sequencing grade) was purchased from Roche Diagnostics (Lewes, UK) and lateral flow immunostrips specific for CP4 EPSPS were from Biofords Sari (Evry, France). GM and non-GM soybean and maize seeds were obtained from Monsanto and distributed by Herbiseed (Twyford, UK).
  • GM and non-GM soybean and maize seeds were ground into a fine homogenous powder using a mechanical grinder.
  • GM soybean at levels of 50 % and 0.9 % (w/w) was added to non-GM soybean powder.
  • the soybean and maize mixtures were then mixed with 30 ml of cold acetone for 45 min and centrifuged at 4,000 g for 10 min at 4 0 C. The acetone wash was repeated 4 times to remove any lipid from the initial mixture.
  • the soybean and maize samples were left to dry overnight at 4 0 C under vacuum.
  • Extraction buffer (40 ml, 50 mM Tris-HCl pH 8, 5 mM DTT, 1 mM EDTA, 1 mM EGTA) was added to the dried soybean and maize pellet and mixed for 2.5 h at 4 0 C. Each sample was then centrifuged at 4,000 g for 1 h at 4 0 C, the pellet was discarded, resulting in a soluble, dilapidated crude extract.
  • the first chromatographic step in the purification procedure utilized a HiLoad 26/60 SuperdexTM 75 gel filtration column (selectivity range 30 to 70 kDa). An aliquot (13 ml) of the reconstituted protein pellet was loaded onto the column. 50 mM Tris-HCl pH 8, 2 mM DTT was used as the mobile phase, at a flow rate of 0.7 ml/min and 7 ml fractions were collected. The protein elution profile was monitored by measuring absorbance at 280 nm. In addition, a Bradford protein assay was performed on each gel filtration fraction according to manufacturer's instructions.
  • a strong anion exchange resin Q Sepharose in a column mode (10 x 2 cm) was used as the second chromatography step.
  • Gel filtration fractions 25 to 29 (GM soya) or 22 to 25 (GM maize) were combined, loaded onto the anion exchange column and washed with 50 ml of 50 mM Tris-HCl pH 8, 2 mM DTT.
  • the proteins were eluted using a linear gradient at a flow rate of 1 ml/min: mobile phase A (50 mM Tris-HCl pH 8, 2 mM DTT) and mobile phase B (50 mM Tris-HCl pH 8, 2 mM DTT, 400 mM NaCl).
  • the protein elution profile was monitored by UV absorbance measurements as described above.
  • Lyophilized gel filtration and anion exchange fractions were reconstituted with 50 ⁇ l and 20 ⁇ l, respectively of 2 X treatment buffer (125 niM Tris-HCl, 4 % SDS, 20 % glycerol, 200 mM DTT, 0.02 % bromophenol blue, pH 6.8), then boiled for 3 min. Aliquots of 6 ⁇ l (gel filtration fractions) and 10 ⁇ l or 20 ⁇ l from GM soya or maize (anion exchange fractions) were loaded onto a 10 % SDS-PAGE and a constant current of 15 mA was applied. The Sigma silver stain kit was employed to stain the gels according to the manufacturer's recommendations.
  • Gel bands were excised at the expected molecular weight of CP4 EPSPS (47 kDa) and destained as described by the manufacturer.
  • the washed gel pieces were mixed with 25 ⁇ l 10 mM DTT (in 25 mM ammonium bicarbonate, pH 8) and incubated at 56 0 C for 1 h.
  • the DTT solution was removed, 25 ⁇ l 55 mM iodoacetamide (in 25 mM ammonium bicarbonate, pH 8) was added and then incubated at room temperature in the dark for 45 min.
  • the gel pieces were washed three times for 20 min with 50 ⁇ l 50 mM ammonium bicarbonate pH 8, and then dried three times with 50 ⁇ l ACN for 15 min.
  • the dried gel pieces were placed on ice for 10 min and 5 ⁇ l (or as required to cover completely the gel) of 12.5 ng/ ⁇ l trypsin (in 50 mM ammonium bicarbonate, pH 8) was added. They were left on ice for 30 min and subsequently 40 ⁇ l 50 mM ammonium bicarbonate, pH 8 was added.
  • trypsin in 50 mM ammonium bicarbonate, pH 8 was added.
  • aliquots of 25 ng/ ⁇ l GIu-C in 100 mM Tris-HCl, pH 7.8) or 20 ng/ ⁇ l Asp-N (in 100 mM Tris- HCl, pH 8.5) were utilized. The samples were incubated at 37 0 C overnight. The supernatant was then transferred into a clean Eppendorf tube.
  • the gel pieces were mixed with 25 ⁇ l ACN:0.1% TFA (50:50, v/v), sonicated for 15 min to elute the peptides and the supernatants were then combined.
  • the solvent was evaporated in vacuo using a GyroVap GT (Howe, UK) and reconstituted with 7 ⁇ l of HPLC- grade water. Alternatively, a 4 ⁇ l aliquot was added to 0. 9 % by weight GM soya fractions.
  • NanoESI MS and MS/MS experiments were performed on a QSTAR Pulsar i (Applied Biosystems, Warrington, UK) hybrid quadrupole time-of-flight mass spectrometer connected to a nanoLC system (LC Packings, Camberley, UK) using a PepMap reverse phase Cj 8 column (15 cm x 75 ⁇ m i.d., 3 ⁇ m, 100 A).
  • the mobile phase consisted of solvent A (water: ACN 99:1 v/v in 0.1 % FA) and solvent.
  • B water: ACN 5:95 v/v in 0.1 % FA).
  • a Protana nanospray interface and 10 ⁇ m distal coated fused silica PicoTips were used for nanoESI.
  • the instrument was automatically calibrated according to the manufacturer's instructions and collision energy was set automatically to produce optimum fragmentation of the precursor ion.
  • Analyst QS 1.0 sp8 software from Applied Biosystems was employed for data analysis.
  • Quadrupolar ion trap MS was carried out on a LCQ DECA from Thermo Finnigan (Cambridge, United Kingdom).
  • the peptide mass lists obtained by MALDI-TOF MS and nanoESI-QTOF MS were submitted for database searching and compared to predicted sequences in the viridiplantae category of the NCBInr and SwissProt databases. Generally, the mass tolerance was set to 100 ppm; up to one missed cleavage was allowed, carbamidomethylation of Cys was considered as a fixed modification and methionine oxidation as a variable modification.
  • nanoLC- ⁇ anoESI- QTOF MS/MS spectra were submitted to MS/MS ion searches for protein identification.
  • Protein extraction facilitated removal of lipids, carbohydrates and small molecules from soybean seeds.
  • the average protein content in crude extracts from two different batches of GM soybean seeds analyzed in duplicate was 15.2 ⁇ 1.2 mg/ml. Fractionation of the GM soybean proteome was necessary as the transgenic CP4 EPSPS could not be identified directly from crude protein extracts.
  • CP4 EPSPS was also identified by MALDI-TOF MS of digested 47 kDa gel bands from other gel filtration (e.g. 25 and 29; Fig. 2B) and other anion exchange fractions (e.g. 75, 77, 80, 85 and 95; Fig. 2D) from 100 % by weight GM soya. For 50 % by weight GM soya the same analytical strategy as presented above was successful.
  • Reverse phase nanoLC offers an additional separation step at the peptide level, and hence could facilitate the identification of the transgenic protein from more complex mixtures when coupled to a nanoESI-QTOF mass spectrometer.
  • CP4 EPSPS marker peptides were mostly detected as doubly charged ions (i.e. [M+2H] 2+ at m/z 558.29, 679.36, 680.32, 779.92, 823.91, 881.93, 997.48, etc.) by nanoLC-nanoESI-QTOF MS. Subsequently, the parent ions were fragmented and the GM protein was identified by MS/MS ion searches.
  • NanoLC separation prior to QTOF MS of in-solution digested CP4 EPSPS containing anion exchange fraction from 50 % by weight GM soya showed CP4 EPSPS marker peptides, for example [M+2H] 2+ at m/z 558.29 (and its corresponding [M+H] + at m/z 1115.61) and [M+2H] 2+ at m/z 779.92 (Fig. 4A and B).
  • a total of eight tryptic peptides were then fragmented and their MS/MS spectra (Fig. 4C and D) enabled the identification of the transgenic protein through database matching.
  • soya proteins were also identified such as glycinin G2 (4 peptides), Gl (7 peptides) and G4 (2 peptides) precursor, napin-type 2S albumin (7 peptides), stress-induced protein SAM22 (3 peptides) and maturation-associated protein MAT9 (2 peptides).
  • napin-type 2S albumin 7 peptides
  • SAM22 3 peptides
  • maturation-associated protein MAT9 2 peptides
  • Tryptic mass maps from the in-gel digested CP4 EPSPS previously fractionated by gel filtration and/or gel filtration followed by anion exchange could be detected by MALDI and nanoESI.
  • nanoLC-nanoESI-QTOF enabled the identification of CP4 EPSPS from more complex protein mixtures.
  • the current EU threshold level for labeling GM-containing products is ⁇
  • Non-GM soybean seeds were analyzed using an identical analytical approach and, as expected, CP4 EPSPS peptides were not observed.
  • selected masses (ion extract chromatogram TOF MS) from CP4 EPSPS tryptic peptides [M+2H] 2+ at m/z 558.29, 779.93 and 881.93 demonstrated the presence of these markers from a 47 kDa digested band of anion exchange combined fractions 82 and 86 from 0.9 % by weight GM soya.
  • these CP4 EPSPS peptides were absent when the same combined fractions from non-GM soybean were analyzed (Fig. 5A-C).
  • GM maize CP4 EPSPS enrichment and MS detection
  • CP4 EPSPS was also identified in 100 % by weight GM maize seeds employing the above strategies.
  • tryptic peptide maps of CP4 EPSPS were obtained from 47 kDa SDS-PAGE bands of gel filtration and anion exchange fractions from 100 % by weight GM maize by MALDI TOF MS that enabled its identification by database searching (Fig. 7). Maize globulin-2 -precursor but not the transgenic protein was detected when a 47 kDa band from the crude protein extract was subjected to peptide mass mapping. CP4 EPSPS peptides from 100 % by weight GM maize were also detected by nanoLC-nanoESI QTOF MS/MS . The CP4
  • EPSPS peptide maps generated from GM maize and GM soya seeds were similar and generally the majority of marker peptides were found in both GM crops.
  • Total sequence coverage of CP4 EPSPS from GM maize was 71 % compared to 75 % from GM soya as reported above, using trypsin, endoproteinase Asp-N and GIu-C.
  • GM and non-GM soyabean seeds were ground into a fine homogenous powder using a mechanical grinder.
  • GM soya at levels of 5, 2, 0.9 and 0.5 % (w/w) was added to non-GM soya powder.
  • the seeds were delipidated using acetone washes. 7 g of the GM soya mixtures were then extracted using 40 ml of 5OmM TEAB, pH 8.5 was employed instead of Tris-HCl.
  • each soya extract was divided into four tubes that generally contained 6 ml of protein solution each. 6 volumes of cold acetone were slowly added to each tube and the crude extracts were mixed for 4 h at 4 0 C to precipitate proteins. The samples were then centrifuged at 4,000 g for 15 min at 4 0 C, and the supernatant was discarded. Subsequently, protein pellets were reconstituted as follows: each protein pellet was mixed with 5 ml of 50 mM TEAB, pH 8.5, vortex mixed for 30 s, sonicated for 5 min and finally centrifuged at 4,000 g 15 min. The supernatants were transferred into a clean tube and the remaining protein pellet was subjected to the same procedure twice more. The four reconstituted pellets from each GM soya preparation were combined in a single tube. Small insoluble pellets were still present in all reconstituted samples, which most likely contain hydrophobic and membrane proteins.
  • Protein separation was carried out using SDS-PAGE as described above. In these experiments the lyophilized samples were reconstituted with 50 ⁇ l of 2 X treatment buffer and 10 ⁇ l were loaded on the gel. Reduction, alkylation and in gel digestion of GM soya samples was performed as described above except that 400 ftnol of a stable isotope labelled peptide was added along with trypsin.
  • the synthetic peptide L* AGGED V ADLR (L* 13 C) was selected as internal standard since LAGGED V ADLR was a particular intense CP4 EPSPS peptide detected by MS.
  • the mass spectrometer was set scan ions from m/z 400 to m/z 1400 and subsequently ions with intensity level above 10 counts were selected for fragmentation using an IDA experiments.
  • Data was processed by manual integration of the peaks in an extracted chromatogram for both monitored peptides (at m /z 558.3 and 561.3) and the selected window was ⁇ 0.2 m/z.
  • the peak area of the native peptide was divided by the peak area of the internal standard.
  • Absolute quantification could be then achieved by multiplying the obtained ratio by the absolute amount of the internal standard (400 fmol) (Refer Figure 8). Furthermore, native/internal standard ratios obtained in each sample could be compared against other GM soya preparations and evaluate against theoretical values, e.g. [5/0.9] % GM should be 5.56.
  • GM soyabean seeds were ground into a fine homogenous powder in an automated freezer mill (Glen Greston Ltd, Stanmore, UK). A 10.0 g sample was then delipidated by acetone washing (5 x 50 ml washings). The resulting protein precipitate was re-suspended in 100 ml of a 50 mM aqueous ammonium bicarbonate solution (pH 8). Trypsin was added to the suspension in a substantial excess to create a protein-to-trypsin ratio of ⁇ 1 : 100. The mixture was left overnight at 37 0 C to permit comprehensive digestion of the transgenic protein.
  • the digested suspension was filtered to separate particulate material from the suspension and the clear supernatant fraction transferred to a 250 ml flask comprising 20 g of a fine polymeric adsorbent selective for LAGGED VADLR.
  • the polymeric adsorbent was assembled from methacrylic acid monomelic units from a LAGGED VADLR template according to the methods generally referred to in Kandimalla VB, Ju H, AnalBioanal Chem (2004) 380, 587-605.
  • the polymeric adsorbent mixture was gently stirred at room temperature overnight to enable binding of LAGGED VADLR present in the digested protein extract to the polymeric adsorbent.
  • Polymeric adsorbent complexed to LAGGEDVADLR was filtered off without washing and dried in vacuo at room temperature. Elution of LAGGEDVADLR was undertaken by rigorous stirring of the dried adsorbent:LAGGED VADLR complex at 37 °C in 100 ml of methanol for 30 minutes. The de-complexed adsorbent was removed from the eluting solvent by filtration and washed with 2 x 50 ml aliquots of the acetonitrile. The supernatant fraction was reduced in vacuo to 100 ml and an excess (0.5 g) of dansyl reagent (Sigma-Aldrich) added with rigorous stirring. The solution was stirred for 2 hours until labelling of LAGGEDVADLR was complete. To remove excess dansyl reagent, the solution underwent HPLC separation with a Cl 8 column.
  • the chromatographed sample of labelled LAGGED VAD LR-dansyl was reduced in vacuo at room temperature to 10 ml and a 1 ml aliquot subjected to UV/Visible spectrometry. From a known value for the extinction co-efficient of LAGGED V ADLR-dansyl derived from synthetic LAGGEDVADLR and its characteristic absorbance wavelength, the concentration of GM protein (ie, EPSPS) present in the GM soyabeans of the example (ie, 10.0 g of starting material) could be easily extrapolated.
  • GM protein ie, EPSPS
  • Predicted tryptic peptides from CP4 EPSPS are sorted by residue number.
  • the experimental CP4 EPSPS matched peptides of a 47 kDa SDS-PAGE band of anion exchange fraction 75 from 50 % by weight GM soybean are also presented.
  • Tryptic peptides were identified by peptide mass fingerprinting (PMF) using MALDI and nanoESI and by MS/MS searches utilizing nanoESI. Matched peptides as a result of one missed cleavage and oxidation are also shown, "oxid" indicates peptide oxidation on methionine.

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Abstract

La présente invention concerne un procédé de détermination de la présence, dans un produit, d'une matière dérivée d'une plante génétiquement modifiée. Le procédé consiste à utiliser un extrait de protéine provenant du produit; à enrichir l'extrait de protéine; à digérer l'extrait de protéine au moyen d'une enzyme; et à détecter la présence ou l'absence d'au moins un peptide marqueur produit par la digestion enzymatique d'une protéine transgénique, ce qui permet de déterminer si la matière dérivée d'une plante génétiquement modifiée est présente dans le produit.
PCT/GB2007/001621 2006-05-02 2007-05-02 Analyse de protéines Ceased WO2007132164A2 (fr)

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WO2014010700A1 (fr) * 2012-07-12 2014-01-16 国立大学法人九州大学 Composé amino, procédé de spectrométrie de masse à sensibilité élevée pour celui-ci et procédé de dosage d'un biomarqueur
CN103698418A (zh) * 2013-11-12 2014-04-02 北京理工大学 植物中转基因蛋白cp4-epsps的定量检测方法
WO2015185030A1 (fr) * 2014-06-06 2015-12-10 Gfl Gesellschaft Für Lebensmittel-Forschung Mbh Procédé de contrôle d'authenticité analytique d'ingrédients dans des aliments en utilisant des anticorps artificiels
WO2015191270A1 (fr) * 2014-06-10 2015-12-17 Dow Agrosciences Llc Analyse quantitative de protéines transgéniques
CN106841458A (zh) * 2017-03-17 2017-06-13 中国肉类食品综合研究中心 液相色谱串联质谱测定肉类食品中植物源性成分的方法
CN109521105A (zh) * 2018-08-03 2019-03-26 西北工业大学 一种用于出土面食文物的鉴定方法

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EP1415137A4 (fr) * 2001-04-17 2007-04-25 Ista S P A Procedes de detection par spectrometrie de masse et quantification de proteines cibles specifiques dans des echantillons biologiques complexes

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CN102458617A (zh) * 2009-06-03 2012-05-16 陶氏益农公司 层叠的转基因蛋白质的多重分析
EP2437869A4 (fr) * 2009-06-03 2013-03-06 Dow Agrosciences Llc Analyse multiplexe de protéine transgénique empilée
WO2010141674A2 (fr) 2009-06-03 2010-12-09 Dow Agrosciences Llc Analyse multiplexe de protéine transgénique empilée
CN102458617B (zh) * 2009-06-03 2015-10-14 陶氏益农公司 层叠的转基因蛋白质的多重分析
AU2010256590B2 (en) * 2009-06-03 2015-05-21 Corteva Agriscience Llc Multiplex analysis of stacked transgenic protein
EP2437869B1 (fr) 2009-06-03 2015-07-22 Dow AgroSciences LLC Analyse multiplexe de protéine transgénique empilée
WO2014010700A1 (fr) * 2012-07-12 2014-01-16 国立大学法人九州大学 Composé amino, procédé de spectrométrie de masse à sensibilité élevée pour celui-ci et procédé de dosage d'un biomarqueur
CN103698418A (zh) * 2013-11-12 2014-04-02 北京理工大学 植物中转基因蛋白cp4-epsps的定量检测方法
WO2015185030A1 (fr) * 2014-06-06 2015-12-10 Gfl Gesellschaft Für Lebensmittel-Forschung Mbh Procédé de contrôle d'authenticité analytique d'ingrédients dans des aliments en utilisant des anticorps artificiels
WO2015191270A1 (fr) * 2014-06-10 2015-12-17 Dow Agrosciences Llc Analyse quantitative de protéines transgéniques
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CN109521105A (zh) * 2018-08-03 2019-03-26 西北工业大学 一种用于出土面食文物的鉴定方法

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