Composition and method for protein detection

Abstract

The present invention relates to peptide biomarkers having specific ionization characteristics for directly quantifying one or more target HPPD proteins in biological samples, including crop samples, by liquid chromatography-coupled tandem mass spectrometry multiple reaction monitoring (MRM). Peptide biomarkers combined with MRM-based methods can be used to quantify a single target protein or multiple target proteins within crops, such as maize, by using selected peptide biomarkers alone or in combination. The present disclosure enables extensive and reliable quantification in various biological matrices, including plant matrices. Additionally, various combinations of peptide biomarkers that can be used to carry out the methods of the present invention are provided.

Description

Technology Field

[0001] Reference to the list of electronically submitted sequences

[0002] An official copy of the sequence list was created on April 20, 2020, and was electronically submitted via EFS-Web as a sequence list in ASCII format with a size of 1 kilobyte and a filename of "81875-WO-REG-ORG-P-1_SeqList.txt", and was filed together with the specification. The sequence list contained in this ASCII format document is part of the specification, and the entirety thereof is incorporated herein by reference.

[0003] Technology field

[0004] The present invention generally relates to the use of mass spectrometry for selectively detecting, quantifying, and characterizing target proteins in complex biological samples. Background Technology

[0005] Immunoassays, such as enzyme-linked immunosorbent assays (ELISA), are currently the preferred method in agriculture for the detection and quantification of proteins introduced through genetic modification or endogenous proteins in plants. A key component of an immunoassay is an antibody that possesses specificity for the target protein (antigen). Immunoassays can be highly specific, and often require only simple preparation of the sample before analysis. Furthermore, immunoassays can be used qualitatively or quantitatively over a wide range of concentrations. Typically, an immunoassay requires individual testing for each protein of interest. Antibodies can be polyclonal, which is elevated in animals, or monoclonal, which is produced by cell culture. Due to their nature, a mixture of polyclonal antibodies will possess multiple recognition epitopes, which can increase sensitivity but also potentially reduce specificity as the likelihood of sequence and structural homology with other proteins increases depending on the number of multiple antibody paratopes present. Monoclonal antibodies offer some advantages over polyclonal antibodies because they exhibit uniform affinity and specificity for a single epitope or epitope and can be produced in vast quantities. However, there are inherent characteristics of all antibodies that limit their use in more demanding applications, such as the selective detection and quantification of a single protein in complex mixtures of similar transgenic or endogenous proteins. Additionally, both polyclonal and monoclonal antibodies may require additional purification steps to improve sensitivity and reduce background in assays. Furthermore, ELISA systems may fail to detect subtle changes in target proteins that could dramatically affect their physical and biological properties. For example, antibodies may fail to recognize specific forms of proteins or peptides that have been altered by post-translational modifications such as phosphorylation or glycosylation, morphologically masked, or modified by partial degradation.Identifying these modifications is essential because changes in the physical and biological properties of these proteins can play a significant role in their enzymatic, clinical, or other biological activities. These changes can limit the reliability and utility of ELISA-based quantification methods.

[0006] Currently, the performance of effective identification and / or quantification of proteins in crops depends on the accuracy of immunoassays. The successful development of immunoassays depends on the specific characteristics of the antigens used in antibody development, namely the size, hydrophobicity, and tertiary structure of the antigens, as well as the quality and accuracy of the antibodies. The specificity of the antibodies must be carefully verified to account for any cross-reactions with similar substances that could lead to false-positive results. A current problem in the industry is that many antibodies in commercially available test kits do not distinguish between similar proteins among various proteins in different crops.

[0007] Mass Spectrometry (MS) provides an alternative platform that overcomes many of the limitations of ELISA for protein analysis. The field of MS-based analysis has achieved significant advancements in targeted protein analysis, such as Multiple Reaction Monitoring (MRM) using electrospray liquid chromatography combined with tandem mass spectrometry (LC-MS / MS). The fundamental concept is that proteins can be quantified by measuring their specific constituent peptides (surrogate peptides) after protein degradation. Data acquisition for selected peptides alone enables measurements with higher precision, sensitivity, and throughput. Protein quantification via MRM-based measurement of surrogate peptides is the fastest-growing MS application in protein analysis. MRM-based protein assays offer two powerful advantages over immunoassay-based assays: first, the ability to systematically organize specific assays for virtually any protein without the use of antibodies; and second, the ability of targeted MS analysis to perform multiplexed analysis of multiple peptides in a single assay. Furthermore, MRM is a direct assay, whereas immunoassay-based assays are indirect. Immuno-based assays rely on binding assays consisting of a ligation reagent that can be immobilized on a solid phase, along with a detection reagent that specifically binds and generates a signal that can be appropriately quantified using an enzyme.

[0008] Therefore, there is a continuing need to identify surrogate peptides that possess all the biochemical characteristics necessary to function in MRM-based assays and have the additional characteristic of being absolutely specific to target proteins that may contain a high proportion of overlapping amino acid sequences (i.e., one or more of the transition states of the surrogate peptide can clearly distinguish two closely related target proteins across multiple complex matrices without interference). These selective surrogate peptides and their transition states must be able to distinguish target proteins that are similar to each other or similar to transgenic proteins in transgenic crops.

[0009] The present invention provides labeled surrogate peptides and their respective transition ions that are useful for selectively detecting or quantifying target proteins present in a complex biological matrix using mass spectrometry. The present invention further provides a method and system for selectively detecting or quantifying target HPPD proteins in a complex biological matrix using labeled surrogate peptides and transition ions.

[0010] In one aspect of the present invention, internal standard peptide markers are designed through empirical analysis and silico-degradation analysis; and are chemically synthesized from heavy amino acid residues or genetically synthesized by expressing a synthetic gene in the presence of stable isotope-labeled amino acid(s) or metabolic intermediates. In certain embodiments, internal standards may be individually characterized by mass spectrometry analysis (MS), including tandem mass spectrometry (MS / MS) analysis, more specifically, liquid chromatography-coupled tandem mass spectrometry (LC-MS / MS) analysis. After characterization, pre-selected peptide parameters of the peptides, such as the single isotope mass of each peptide, its surrogate charge state, surrogate m / z value, m / z transition ion, and the ion type of each transition ion, may be collected. Other considerations include peptide size optimization, prevention of post-translational modification, prevention of process-induced modification, and prevention of a high rate of missing protease cleavage.

[0011] In one embodiment, the present invention relates to a mixture of proteins in one or more biological samples from one or more crops. p-The present invention provides a labeled surrogate peptide that functions in a mass spectroscopic assay to selectively detect or quantify hydroxyphenylpyurbate deoxygenase (HPPD) protein, wherein the surrogate peptide comprises a label and an amino acid sequence selected from the group consisting of GNFSELFK (SEQ ID NO:1) and GNFSQLFK (SEQ ID NO:2). In some embodiments, the labeled surrogate peptide is labeled by incorporating a stable isotope label (SIL) amino acid. In other embodiments, the SIL amino acid is lysine, isoleucine, valine, or arginine. In other embodiments, the plant is barley, rice, soybean, wheat, oat, or maize. In yet another embodiment, the crop is barley, rice, soybean, wheat, or rice, and the surrogate peptide comprises the amino acid sequence of the label and SEQ ID NO:1. In another embodiment, the crop is maize, and the surrogate peptide includes the label and the amino acid sequence of SEQ ID NO:2.

[0012] In one embodiment, the present invention provides an assay cassette comprising at least two label surrogate peptides of claim 1.

[0013] In one embodiment, the present invention provides a method for simultaneously detecting or quantifying one or more target HPPD proteins in a complex biological sample from a crop comprising a mixture of target proteins and non-target proteins, the method comprising the following steps: (a) obtaining a biological sample from a crop; (b) extracting proteins from the biological sample to produce an extract comprising a mixture of proteins; (c) reducing the amount of insoluble proteins in the extract of step b to produce an extract of concentrated soluble proteins; (d) degrading the soluble proteins in the extract of step c to produce an extract comprising peptide fragments, wherein the peptide fragments comprise at least one surrogate peptide specific to the target protein; (f) concentrating the peptide fragments in the extract of step d; (f) adding one or more labeled surrogate peptides of the present invention, wherein each labeled surrogate peptide has the same amino acid sequence as each surrogate peptide of the target protein, and the number of added labeled surrogate peptides is equal to the number of target proteins in the mixture; (g) a step of concentrating the surrogate peptide and the labeled surrogate peptide by reducing the amount of non-surrogate peptide in the mixture; (h) a step of dissolving the mixture of peptide fragments from step g through liquid chromatography; (i) a step of analyzing the mixture of peptide fragments generated from step h through mass spectrometry, wherein the detection of a transion fragment of the labeled surrogate peptide indicates the presence of the target protein from which the surrogate peptide originated; and, optionally, (j) a step of calculating the amount of the target protein in a biological sample by comparing the mass spectrometry signal generated from the transion fragment of step i with the mass spectrometry signal generated by the transduction. In some embodiments, the crop is barley, rice, soybean, wheat, or paddy, and the surrogate peptide comprises the label and the amino acid sequence of SEQ ID NO:1.In another embodiment, the crop is maize, and the surrogate peptide comprises the label and the amino acid sequence of SEQ ID NO:2. In another embodiment, the peptide is labeled by the incorporation of a stable isotope label (SIL) amino acid. In yet another embodiment, the SIL amino acid is lysine, isoleucine, valine, or arginine.

[0014] A number of different combinations of surrogate peptides can be simultaneously monitored and quantified by MRM assays with one or more of the specific surrogate peptides from the HPPD proteins of the present invention, thereby providing means to measure the total amount of each of such proteins in a given protein preparation obtained from a biological sample by mass spectrometry. Together with MRM-based assays, these peptides have numerous applications, including quantitative peptide / protein assays for determining expression levels in crops at different growth stages; for determining expression levels in different crop tissues and organs, including but not limited to leaf tissue, seeds and grains, pollen and root tissues; for determining potential exposure levels for regulatory risk assessments; and for determining different levels of proteins in food processing, comparative, and generational studies. In the broadest sense, these unique surrogate peptides for seven proteins can be used in combination with MRM assays for numerous applications, including agricultural applications, biological equivalence testing, biomarkers, diagnostics, discovery, food, environment, and therapeutic monitoring in all types of biological and non-biological matrices. In some embodiments of the present invention, a test cassette comprising one or more label surrogate peptides of the present invention is provided, which enables simultaneous and selective detection or quantification of any one or more target proteins of the present invention.

[0015] The present invention also provides a method for selectively detecting or quantifying a target HPPD protein within a complex biological matrix, such as a biological sample from a crop. This method comprises the steps of: obtaining a sample from a crop, e.g., a leaf, seed or grain, pollen, or root; extracting a protein from the plant sample; concentrating a target protein pool by reducing the amount of insoluble protein in the extract; digesting the soluble protein in the extract with a selected enzyme, e.g., trypsin, to produce an extract containing peptide fragments, wherein the peptide fragments contain at least one surrogate peptide specific to each target protein; adding a assay cassette of SIL peptides for specifically detecting the target protein, wherein each labeled surrogate peptide has the same amino acid sequence as each surrogate peptide of the target protein, and the number of added labeled surrogate peptides is equal to the number of target proteins in the mixture; concentrating the surrogate peptides and labeled surrogate peptides by reducing the amount of non-surrogate peptides in the mixture; and dissolving the mixture of peptide fragments using liquid chromatography. The method comprises the step of analyzing a mixture of peptide fragments using mass spectrometry, wherein the detection of a transition ion fragment of a labeled surrogate peptide indicates the presence of the target protein from which the surrogate peptide originated; and, optionally, the step of calculating the amount of the target protein in a biological sample by comparing the mass spectrometric signal generated from the transition ion fragment with the mass spectrometric signal generated by the transition ion of the labeled surrogate peptide. Each SIL surrogate peptide derived from the protein of the present invention possesses a unique transition ion during mass spectrometry-based multiple reaction monitoring (MRM) assays. As such, these peptides will generate selective MS ions that cause different degrees of ionization due to slight changes in collision energy. For example, triple quadrupole MS can be used to generate peptide-specific high m / z ions.Consequently, the method of the present invention can provide a selective advantage of reducing the endogenous background compared to the use of lower m / z intensity ion markers that may be known in the art.

[0016] The present invention further provides a system for high-throughput detection or quantification of a target protein. Such a system comprises a cassette of pre-designed labeled surrogate peptides specific to the target protein; and one or more mass spectrometers.

[0017] Various objects, features, aspects, and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the present invention, together with the accompanying drawings and sequence list.

[0018] A brief explanation of the hierarchy

[0019] SEQ ID NO:1 is the amino acid sequence of a stable isotope-labeled surrogate peptide for the selective detection and quantification of HPPD proteins in barley, rice, soybeans, wheat, and oats.

[0020] SEQ ID NO:2 is the amino acid sequence of a stable isotope-labeled surrogate peptide for the selective detection and quantification of HPPD protein in maze. Specific details for implementing the invention

[0021] This description does not constitute a detailed catalog of all different ways in which the present invention may be implemented, or of all features that may be added to the present invention. For example, features illustrated in relation to one embodiment may be introduced into another embodiment, or features illustrated in relation to a specific embodiment may be omitted from such embodiment. Accordingly, the present invention takes into account that any feature or combination of features described herein may be excluded or omitted in some embodiments of the present invention. Furthermore, numerous variations and additions to the various embodiments proposed herein that do not depart from the present invention will be apparent to those skilled in the art in light of this disclosure. Accordingly, the following description is intended to illustrate some specific embodiments of the present invention and is not intended to fully specify all substitutions, combinations, and variations thereof.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the invention. Furthermore, it should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention. General reference to the present invention includes the following documents: Alwine et al. (1977) Proc. Nat. Acad. Sci. 74:5350-54; Baldwin (2004) Mol. Cell. Proteomics 3(1):1-9; Can and Annan (1997) Overview of peptide and protein analysis by mass spectrometry. In: Current Protocols in Molecular Biology, edited by Ausubel, et al. New York: Wiley, p. 10.21.1-10.21.27; Chang et al. (2000) Plant Physiol. 122(2):295-317; Domon and Aebersold (2006) Science 312(5771):212-17; Nain et al. (2005) Plant Mol. Biol. Rep. 23:59-65; Patterson (1998) Protein identification and characterization by mass spectrometry. In: Current Protocols in Molecular Biology, edited by Ausubel, et al. New York: Wiley, p. 10.22.1-10.22.24; Paterson and Aebersold (1995) Electrophoresis 16: 1791-1814; Rajagopal and Ahern (2001) Science 294(5551):2571-73; Sesikeran and Vasanthi (2008) Asia Pac. J. Clin. Nutr. 17 Suppl. 1:241-44; and Toplak et al. (2004) Plant Mol. Biol. Rep. 22:237-50.

[0023] definition

[0024] As used herein and in the appended claims, the singular indefinite and definite articles may mean one or more than one. Thus, for example, a reference to "plant" may mean a single plant or multiple plants.

[0025] As used herein, "and / or" refers to and encompasses all possible combinations of one or more of the relevant enumerated items, as well as the absence of such combinations when interpreted as an alternative ("or").

[0026] The term "about" is used herein to mean approximately, nearly, about, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the presented numerical value. Generally, the term "about" is used herein to modify a value higher or lower than the specified value by a variation of 20%, preferably 10% or more or less (higher or lower). With respect to temperature, the term "about" means ± 1°C, preferably ± 0.5°C. When the term "about" is used in the context of the present invention (e.g., in combination with temperature or molecular weight values), an exact value (i.e., no "about") is preferred.

[0027] The terms “comprises” and / or “comprising”, as used herein, specify the presence of the specified features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0028] The transition phrases "consisting essentially of" and their grammatical variations used herein shall be interpreted as encompassing the scope of the claims, including the specific materials or steps listed in the claims and those that do not substantially alter the basic and novel feature(s) of the claimed invention. Accordingly, the term "consisting essentially of" is not to be interpreted as equivalent to "comprising" when used in the claims of the present invention.

[0029] As used herein, the term "transgenic event" refers to a recombinant plant produced by the transformation and regeneration of a single plant cell containing heterologous DNA, for example, by an expression cassette containing the gene of interest. The term "event" refers to the offspring of the original transformant and / or the transformant containing heterologous DNA. The term "event" also refers to offspring produced by sexual crossing between a transformant and another maize line. Even after repeated backcrossings against the recurrent parent, the inserted DNA and adjacent DNA from the transformed parent are present in the offspring of the crossing at the same chromosomal location. Normally, the transformation of plant tissues generates multiple events, each of which represents the insertion of a DNA construct at a different location in the plant cell genome. A specific event is selected based on the expression of the transgene or other desirable characteristics. A non-limiting example of such a transgenic event of the present invention comprises the cry1Ab and pat genes and the “event Bt11” (also “Bt11 event” or simply “Bt11”) described in US No. 6114608, eCry3."Event 5307" (also "Event 5307" or simply "5307") as described in US heading No. 8466346, comprising 1Ab and PMI genes; "Event MIR604" (also "Event MIR604" or simply "MIR604") as described in US heading No. 7361813, comprising mCry3A and PMI genes; "Event MIR162" (also "Event MIR162" or simply "MIR162") as described in US heading No. 8232456, comprising Vip3A and PMI genes; "Event GA21" (also "Event GA21" or simply "GA21") as described in US heading No. 6566587, comprising dmEPSPS genes; "Event 3272" (also "Event 3272" or simply "3272") as described in US heading No. 7635799, comprising alpha-amylase 797E and PMI genes; and Cry1Ab "Event MON810" (also "Event MON810" or simply "MON810"), Cry1A.105 and Cry2Ab genes as described in US heading 6713259, "Event MON89034" (also "Event MON89034" or simply "MON89034"), as described in US heading 8062840, Cry1F and PAT genes, "Event TC1507" (also "Event TC1507" or simply "TC1507"), as described in US heading 7288643, Cry34 / Cry35 and PAT genes, "Event DAS59122" (also "Event DAS59122" or simply "DAS59122"), as described in US heading 7323556, and "Event" as described in US heading 9790561, comprising Cry1F, Cry34 / Cry35 and PAT genes It includes DP4114 (also "DP4114 event" or simply "DP4114").

[0030] The term "isolated" nucleic acid molecule, polynucleotide, or toxin is a nucleic acid molecule, polynucleotide, or toxic protein that no longer exists in its natural environment. The isolated nucleic acid molecule, polynucleotide, or toxin of the present invention may exist in a purified form or in a recombinant host, such as a transgenic bacterial cell or a transgenic plant.

[0031] The general term "mass spectrometry" as used herein refers to any suitable mass spectrometry method, apparatus, or configuration, including, for example, electrospray ionization (ESI), matrix-assisted laser desorption / ionization (MALDI) MS, MALDI-time-of-flight (TOF) MS, atmospheric pressure (AP) MALDI MS, vacuum MALDI MS, tandem MS, or any combination thereof. A mass spectrometry apparatus measures the molecular mass of a molecule (as a function of the molecule's mass-to-charge ratio) by measuring the flight path of a molecule through a series of magnetic and electric fields. The mass-to-charge ratio is a widely used physical quantity in the electrodynamics of charged particles. The mass-to-charge ratio of a particular peptide can be calculated a priori by those skilled in the art. Two particles with different mass-to-charge ratios will not travel along the same path in a vacuum when the same electric and magnetic fields are applied. The present invention, in particular, involves the use of high-performance liquid chromatography (HPLC) followed by tandem MS analysis of peptides. In "tandem mass spectrometry," the surrogate peptide can be filtered from the MS instrument, and the surrogate peptide is subsequently fragmented to obtain one or more "transition ions," which are analyzed in a second MS procedure (detected and / or quantified).

[0032] A detailed overview of the methodology and apparatus of mass spectrometry can be found in the following references incorporated herein by reference: Can and Annan (1997) Overview of peptide and protein analysis by mass spectrometry. In: Current Protocols in Molecular Biology, edited by Ausubel, et al. New York: Wiley, p. 10.21.1-10.21.27; Paterson and Aebersold (1995) Electrophoresis 16: 1791-1814; Patterson (1998) Protein identification and characterization by mass spectrometry. In: Current Protocols in Molecular Biology, edited by Ausubel, et al. New York: Wiley, p. 10.22.1-10.22.24; and Domon and Aebersold (2006) Science 312(5771):212-17.

[0033] Peptides are short polymers formed from the linking of alpha-amino acids in a defined sequence. Peptides can also be produced by the degradation of polypeptides, for example, proteins, using proteases.

[0034] "Plants" are any plants at any stage of development, in particular, seed plants.

[0035] A "plant cell" is the structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or, for example, part of a highly organized unit such as a plant tissue, a plant organ, or an entire plant.

[0036] "Plant cell culture" refers to a culture of plant units at various developmental stages, such as, for example, protoplasts, cell culture cells, cells among plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos.

[0037] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, eggs, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0038] "Plant organs" are unique, visibly structured, and differentiated parts of a plant, such as roots, stems, leaves, flower buds, or embryos.

[0039] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. It includes any tissue of a plant within a plant body or culture. This term includes, but is not limited to, whole plants, plant organs, plant seeds, plant cultures, and any group of plant cells organized into structural and / or functional units. The use of this term with, or in the absence of, any specific form of plant tissue as listed above or otherwise included by this definition does not imply the exclusion of any other type of plant tissue.

[0040] As used herein, the term “surrogate peptide” refers to a peptide derived from a target protein through proteolysis, e.g., trypsin degradation, which is combined with the surrogate peptide when the target protein is present in the presence of one or more other proteins and / or transgenic proteins in a complex biological matrix, e.g., a sample from a crop, to differentially detect and / or quantify the target protein and to generate one or more transition ions that do not detect and / or quantify one or more other proteins or transgenic proteins in the biological matrix. The “surrogate peptide” may also be referred to as a “signature peptide” for the target protein. For example, the HPPD surrogate peptide of the present invention is combined with the HPPD surrogate peptide when the HPPD protein is present in the presence of one or more non-HPPD proteins to generate one or more transition ions that differentially detect and / or quantify the target HPPD protein in a complex biological matrix. According to an embodiment of the present invention, two or more labeled surrogate peptides of the present invention can be used simultaneously in a mass spectrometry assay to detect and / or quantify two or more target proteins in a complex biological matrix.

[0041] "Labeled surrogate peptide" is a non-naturally occurring surrogate peptide that is labeled for the easy detection of the surrogate peptide in mass spectrometry assays. For example, the label may be a stable isotope-labeled amino acid (SIL), such as lysine, isoleucine, valine, or arginine. Thus, the SIL-labeled surrogate peptide has the same amino acid sequence as the non-labeled surrogate peptide, except that one or more amino acids of the surrogate peptide are labeled with a heavy isotope. For example, as described herein, the surrogate peptide GNFSELFK (SEQ ID NO:1) is labeled with heavy lysine (K) and may be denoted as GNFSELFK [C13N15-K], etc.

[0042] As used herein, the terms "stacked" or "stacking" refer to the presence of multiple heterogeneous polynucleotides, transgenic proteins, or transgenic events incorporated into the plant genome.

[0043] As used herein, "target protein" refers to a protein intended to be selectively detected and / or quantified by a labeled surrogate peptide when the target protein is present in a complex biological matrix.

[0044] Nucleotides are denoted herein by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise denoted by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0045] The present invention comprises compositions, methods, and systems useful for performing mass spectrometry for the differential detection and / or quantification of one or more target HPPD proteins in complex biological samples derived from crops, for example, biological samples from leaves, stems, roots, pollen, and seeds of one or more crops, each comprising a mixture of target and non-target proteins that may differently affect the mass spectrometry calibration results.

[0046] The accuracy of target protein quantification by mass spectrometry multiple response monitoring assays (MRM) depends entirely on the selection of appropriate surrogate peptides and the ability of surrogate peptide / transition ion combinations to distinguish target proteins. Multiple different combinations of surrogate peptides of the present invention can be simultaneously monitored and detected by MRM assays with one or more specific peptides from HPPD proteins, thereby providing a means to identify and quantify each target HPPD protein within a given biological sample by mass spectrometry. The available surrogate peptides constituting the cassette can be analyzed alone or in any combination in a single MRM assay, or can be analyzed in multiple MRM assays.

[0047] In combination with MRM-based assays, the surrogate peptides of the present invention have numerous applications, including quantitative peptide / protein assays for determining expression levels at different growth stages, determining potential exposure levels for environmental risk assessments, determining different levels of target proteins in food processing, determining expression levels in comparative studies, and comparing expression levels in intergenerational studies. In the broadest sense, these unique surrogate peptides for target proteins can be used in combination with MRM assays to monitor or quantify herbicide resistance traits, which may be a breeding stack of crop or transgenic events, or multiple transgenic events, within specific tissues (i.e., leaves, roots, kernels, pollen).

[0048] MRM-based assays can quantify or measure the relative or absolute levels of specific surrogate peptides from HPPD proteins. The relative quantitative levels of these proteins can be determined by MRM assays by comparing signature peak areas. The relative levels of individual HPPD surrogate peptides can be quantified from different samples or tissue types. Generally, relative quantitative levels are determined by comparing the peptide abundance in MRM measurements with a stable isotope-labeled (SIL) synthetic peptide analog as an internal standard for each target surrogate peptide. Unlike what is typically taught in the art, SIL peptides [ 13 C6 15 N2] Ricin or [ 13 C6 15[N4] It is labeled by the incorporation of arginine, but may also contain other amino acids such as isoleucine and valine. The SIL standard must be of high purity and quantitatively standardized by amino acid analysis. Unlike what is typically taught in the art, the SIL of the present invention is spiked into the sample immediately after protein degradation, thereby serving to correct for subsequent analysis steps. The SIL is co-exposed with an unlabeled surrogate peptide in liquid chromatography separation and exhibits the same MS / MS fragmentation pattern, differing only in mass due to isotopic labeling. Due to the mass change of both the generated labeled surrogate peptide and the resulting ion, it is possible for a mass spectrometer to distinguish between the unlabeled peptide and the labeled peptide. Since complex peptide degradation products often contain multiple sets of co-exposed transitions that can be mistaken for the target peptide, co-exposition with the isotopic-labeled standard provides the best protection against false-positive quantification and the identification of the correct signal. Since spiked SIL standards of known concentrations are spiked into each sample, the relative quantitative amount of each corresponding surrogate peptide from different target proteins can be determined for HPPD proteins. Because the relative quantification of individual peptides, or peptides, can be performed in proportion to the amount of another peptide, or peptides, within or between samples, it is possible to determine the relative amount of the peptide present by determining whether the peak areas are relative to each other within the biological samples. Relative quantitative data inferred from individual signature peak areas between different samples is generally normalized to the amount of protein analyzed per sample. Relative quantification can be performed across multiple samples simultaneously across multiple peptides from multiple proteins in a single sample and / or across multiple samples to gain further insight into the relative protein amount of one peptide / protein to another peptide / protein.

[0049] Absolute quantitative levels can be determined for HPPD by an MRM-based assay by comparing the signature peak area of ​​individual surrogate peptides from corresponding proteins in a single biological sample with known amounts of one or more internal standards in the sample. This can be achieved by spiking these proteins at known concentrations into a negative control matrix that does not contain the target proteins. The multiple-reaction monitoring (MRM) assay comprises the steps of: weighing a non-target sample containing the correct spiked concentration of the target protein; extracting and homogenizing the sample in a lysis buffer; centrifuging the sample into distinct soluble and abundant insoluble proteins to reduce the complexity of the extraction; The method comprises the steps of: digesting a soluble protein sample with trypsin (tissue or biological samples may be treated with one or more proteases, including but not limited to trypsin, chymotrypsin, pepsin, endoproteinases Asp-N and Lys-C, for a predetermined time to ensure the sample is properly digested); centrifuging the sample and adding a fixed concentration of SIL peptide (SIL is used as an indicator in absolute quantification); desalting by solid-phase extraction using cation exchange to minimize matrix effects or interference and reduce ion inhibition; and analyzing the sample by liquid chromatography coupled with tandem mass spectrometry. Typically, an ion trap mass spectrometer, or another form of mass spectrometer capable of performing global profiling, is operated for analysis to identify as many peptides as possible from a single complex protein / peptide lysate. Although MRM-based assays can be developed and performed on any type of mass spectrometer, the instrument platform most advantageous for MRM assays is commonly considered to be the triple quadrupole instrument platform. The surrogate peptides and SILs unique to the 7 proteins of interest are measured by LC-MS / MS. The peak area ratio (peak area of ​​the surrogate peptide / peak area of ​​the corresponding SIL peptide) is determined for each peptide of interest.The concentrations of the seven proteins of interest are inversely calculated from the calibration curve using the peak area ratio. Absolute quantification can be performed across multiple peptides (enabling simultaneous quantitative measurement of multiple proteins in a single sample) and / or across multiple samples (to gain insight into absolute protein amounts in individual biological samples or large sample sets).

[0050] In some embodiments, the present invention relates to a mixture of proteins in one or more biological samples from one or more crops. p- As a labeled surrogate peptide functioning in a mass spectroscopic assay to selectively detect or quantify hydroxyphenylpyurbate deoxygenase (HPPD) protein, the surrogate peptide comprises a label and an amino acid sequence selected from the group consisting of GNFSELFK (SEQ ID NO:1) and GNFSQLFK (SEQ ID NO:2). In some embodiments, the labeled surrogate peptide is labeled by the incorporation of a stable isotope label (SIL) amino acid. In other embodiments, the SIL amino acid is lysine, isoleucine, valine, or arginine. In other embodiments, the crop is barley, rice, soybean, wheat, oats, or maize. In yet another embodiment, the crop is barley, rice, soybean, wheat, or rice, and the surrogate peptide comprises the amino acid sequence of the label and SEQ ID NO:1. In another embodiment, the crop is a maize, and the surrogate peptide contains the amino acid sequence of label and SEQ ID NO:2.

[0051] In some embodiments, the present invention comprises a test cassette comprising at least two labeled surrogate peptides comprising amino acid sequences selected from the group consisting of GNFSELFK (SEQ ID NO:1) and GNFSQLFK (SEQ ID NO:2).

[0052] In some embodiments, the present invention comprises a method for simultaneously detecting or quantifying one or more target HPPD proteins in a complex biological sample from a crop comprising a mixture of target proteins and non-target proteins, the method comprising the following steps: (a) obtaining a biological sample from a crop; (b) extracting proteins from the biological sample to produce an extract comprising a mixture of proteins; (c) reducing the amount of insoluble proteins in the extract of step b to produce an extract of concentrated soluble proteins; (d) degrading the soluble proteins in the extract of step c to produce an extract comprising peptide fragments, wherein the peptide fragments comprise at least one surrogate peptide specific to the target protein; (e) concentrating the peptide fragments in the extract of step d; (f) adding one or more labeled surrogate peptides of the present invention, wherein each labeled surrogate peptide has the same amino acid sequence as each surrogate peptide of the target protein, and the number of added labeled surrogate peptides is equal to the number of target proteins in the mixture; (g) a step of concentrating the surrogate peptide and the labeled surrogate peptide by reducing the amount of non-surrogate peptide in the mixture; (h) a step of dissolving the mixture of peptide fragments from step g through liquid chromatography; (i) a step of analyzing the mixture of peptide fragments generated from step h through mass spectrometry, wherein the detection of a transion fragment of the labeled surrogate peptide indicates the presence of the target protein from which the surrogate peptide originated; and, optionally, (j) a step of calculating the amount of the target protein in a biological sample by comparing the mass spectrometry signal generated from the transion fragment of step i with the mass spectrometry signal generated by the transduction. In some embodiments, the crop is barley, rice, soybean, wheat, or paddy, and the surrogate peptide comprises the label and the amino acid sequence of SEQ ID NO:1.In another embodiment, the crop is maize, and the surrogate peptide contains the label and the amino acid sequence of SEQ ID NO:2. In another embodiment, the peptide is labeled by the incorporation of a stable isotope label (SIL) amino acid. In yet another embodiment, the SIL amino acid is lysine, isoleucine, valine, or arginine.

[0053] In the art, there are numerous literatures proposing various different methods for predicting which surrogate peptide is best for any given target protein, and numerous references, for example, the literature [Mead et al[2009. Mol. Cell. Proteomics 8:696-705] and U.S. Patent No. 8,227,252 propose a simple method for quantifying target proteins using mass spectrometry. However, reliance on these predictive and simple methods can lead to confounding results because unpredictable factors can interfere with mass spectrometry-based assays, resulting in inaccurate quantification and loss of sensitivity. At least one major factor lies in the biological matrix itself. For example, identifying a single transition ion from a surrogate peptide that works equally well with biological samples from leaves, roots, pollen, and seeds from crops is highly unpredictable and difficult. Differences in the chemical composition, pH, or ionic strength of the matrix can affect proteolysis, peptide stability, aggregation, or ionization on MS instruments. Therefore, identifying and empirically testing surrogate peptides and specific surrogate peptide / transition ion combinations across all relevant matrices, particularly those for crops, is essential to overcome the unpredictable nature of these assays. The present invention utilizes a two-step approach to developing a mass spectrometry assay to specifically detect and / or quantify a target protein, comprising: 1) testing and selecting surrogate peptides from a pool of peptides derived from proteolytically cleaved target proteins, testing combinations of SIL surrogate peptides and transfer ion peptides, and selecting combinations that specifically detect and quantify the target protein across all biological samples from any biological matrix, e.g., leaves, roots, pollen, or seeds of crops; and 2) empirically determining appropriate sample preparation methods and mass spectrometer conditions that work for all surrogate peptides and surrogate peptide / transfer ion combinations in any biological matrix, including leaves, roots, pollen, and seeds of crops, particularly maize plants.

[0054] Accordingly, in some embodiments, the present invention comprises a method for simultaneously detecting and / or quantifying one or more target proteins in a complex biological sample from a transgenic plant comprising a mixture of target transgenic proteins and non-transgenic proteins, wherein the method comprises the following steps: a) obtaining a biological sample from a transgenic plant; b) extracting proteins from the biological sample to produce an extract comprising a mixture of proteins; c) reducing the amount of non-transgenic insoluble proteins in the extract of step b to produce an extract of concentrated soluble proteins; d) degrading the soluble proteins in the extract of step c to produce an extract comprising peptide fragments, wherein the peptide fragments comprise at least one non-labeled surrogate peptide specific to each target protein; e) concentrating the peptide fragments in the extract of step d; f) adding one or more labeled surrogate peptides of the present invention, wherein each labeled surrogate peptide has the same amino acid sequence as each non-labeled surrogate peptide derived from the target protein, and the number of added labeled surrogate peptides is equal to the number of target proteins in the mixture; g) a step of concentrating the unlabeled surrogate peptide and the labeled surrogate peptide by reducing the amount of the unlabeled surrogate peptide in the mixture; h) a step of dissolving the peptide fragment mixture from step g through liquid chromatography; i) a step of analyzing the peptide fragment mixture generated from step h through mass spectrometry, wherein the detection of the transion fragment of the unlabeled surrogate peptide indicates the presence of the target protein from which the surrogate peptide originated; and, optionally, j) a step of calculating the amount of the target protein in a biological sample by comparing the mass spectrometry signal generated from the transion fragment of step i with the mass spectrometry signal generated by the transion of the labeled surrogate peptide.

[0055] The following specific examples are included to demonstrate preferred embodiments of the present invention. It should be recognized by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention and may be considered to constitute a preferred method for practicing the invention. However, those skilled in the art should recognize that, in light of this disclosure, many variations may be made in the specific embodiments disclosed and similar or similar results may still be obtained without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may replace the agents described herein while achieving the same or similar results. All such similar substitutions and variations apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined in the appended claims.

[0056] Examples

[0057] Although the present invention has been described in connection with specific embodiments thereof, it will be understood that further modifications of the device of the present invention are possible. This patent application is intended to include any variation, use, or modification of the present invention (generally following the principles of the present invention, falling within the known or customary practices within the art to which the invention pertains, and applicable to the essential features of the present invention prior to description, and including deviations from the present disclosure within the scope of the appended claims).

[0058] All publications and patent applications mentioned herein represent the level of skill of those skilled in the art to which the present invention pertains. All publications and patent applications herein are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated by reference.

[0059] Example 1. Endurance in commercially available crop products using mass spectrometry assay p- Verification of the quantification of hydroxyphenylpyruvate deoxygenase

[0060] The purpose of this study is to investigate various commercial crops (barley, maize, rice, soybean, and wheat seeds, and barley, maize, oat, soybean, and wheat feed) using liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS). p- The objective was to validate a mass spectrometry (MS)-based method for the quantification of hydroxyphenylpyruvate deoxygenase (HPPD) protein. Relative concentrations were determined using surrogate peptides unique to HPPD protein. Prior to implementing the quantitative method in the study, the method was validated for its intended use according to Good Laboratory Practices (GLPS). Method performance parameters, including specificity, linearity, limit of quantification, carryover, precision and accuracy, extraction efficiency, and stability, were evaluated using commercial crop samples.

[0061] Specificity evaluation did not show significant interference with the retention times of unlabeled peptides (mean ratio difference between the two transitions between blank and buffer was less than 30.0%) and stable isotope-labeled (SIL) peptides (SIL signal of QC0 (endogenous) was less than 5.0%) in any commercial crop sample tested. All specificity parameters met the acceptance criteria.

[0062] The lower limit of quantification (LLOQ) for each signature peptide used as a surrogate for the target HPPD protein was determined. The limits of quantification were 0.209 μg / g dry weight (DW) for barley seeds, 0.122 μg / g DW for maize seeds, 0.083 μg / g DW for rice seeds, 0.686 μg / g DW for soybean seeds, 0.301 μg / g DW for wheat seeds, 0.524 μg / g DW for barley feed, 0.326 μg / g DW for maize feed, 0.660 μg / g DW for oat feed, 1.001 μg / g DW for soybean feed, and 0.640 μg / g DW for wheat feed.

[0063] The effective quantification ranges (LLOQ and ULOQ) of HPPD for each commercial crop are summarized in Table 1.

[0064] [Table 1]

[0065] Quantitative range

[0066]

[0067] The within-test and between-test CVs were 25.0% or less for QC0 samples and 20.0% or less for Quality Control (QC) samples across the three concentrations (low, medium, and high), indicating excellent method precision. Similarly, the within-test and between-test bias % was within ±20.0% for low, medium, and high QC samples, indicating excellent method accuracy. Therefore, all method precision and accuracy parameters met the acceptance criteria. The within-test and between-test precision and accuracy of the method are summarized in Table 2.

[0068] [Table 2]

[0069] Range of intra-test and inter-test precision and accuracy for HPPD in commercial crops

[0070]

[0071] The relationship between concentration and detector response was represented using a linear equation with a data weight of 1 / x. Therefore, all established data fell within acceptable limits and were confirmed to be suitable for linearity evaluation.

[0072] The efficiency of the protein extraction method was 59.9% for barley seeds, 69.8% for maize seeds, 73.9% for rice seeds, 64.9% for soybean seeds, 54.3% for wheat seeds, 69.8% for barley feed, 76.3% for maize feed, 78.4% for oat feed, 65.4% for soybean feed, and 77.2% for wheat feed. The CV of the first round of extraction was less than 20.0% for all commercial crops, except for wheat seeds, which had a CV of 27.5%. However, the precision and accuracy results (QC0) for wheat seeds obtained from a single extraction demonstrate that a precision of 20.0% or less was achieved.

[0073] Processing stability (dried extract) was evaluated at -20°C for 6 days. The stability evaluation met the acceptance criteria. CV was 25.0% or less, and the % difference in peak area ratio between stability and Day 0 QC was within ± 25.0% for all commercial crops.

[0074] All evaluated performance parameters met the prescribed acceptance criteria, with the exception of the final extraction percentages for barley and wheat seeds, which were 8.2% and 6.2%, respectively, as well as the precision of the first round extraction for wheat seeds, which was 27.5% CV. In the sample analysis, the concentration of HPPD was determined from a single extraction (first round) and adjusted for extraction efficiency %; since precision of 20.0% or less was achieved for wheat seeds in the precision and accuracy progression, there was no impact on the study results. Based on the results of this study, mass spectrometry-based methods were confirmed to be suitable for the quantification of HPPD proteins in commercial crops according to GLPS.

[0075] The purpose of this study is to investigate various commercial crops (barley, maize, rice, soybean, and wheat seeds, and barley, maize, oat, soybean, and wheat feed) using liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) analysis. p- The objective was to validate a mass spectrometry (MS)-based method for the quantification of hydroxyphenylpyruvate deoxygenase (HPPD) protein. Relative concentrations among different plant species / matrices were determined using surrogate peptides unique to HPPD protein (Table 3). Prior to implementing the quantitative method in the study, the method was validated for its intended use according to Good Laboratory Practices (GLPS). Method performance parameters, including specificity, linearity, limit of quantification, carryover, precision and accuracy, extraction efficiency, and stability, were evaluated using commercial crop samples.

[0076] [Table 3]

[0077] HPPD surrogate peptide

[0078]

[0079] The commercial crop material for this study was a matrix from various plant species used to prepare linearity samples and quality control (QC) samples. Table 4 identifies the sources of the matrix material. These samples were provided by sponsors and stored at a nominal temperature of -80°C ± 10°C until use.

[0080] [Table 4]

[0081] commercial crop material

[0082]

[0083] Standards and QC samples

[0084] Reversal curves were generated to evaluate linearity. A series of eight non-zero standards (STDs) were prepared using commercial crop extracts enriched with SIL peptides at different concentrations, including both LLOQ (STD 1) and ULOQ (STD 8) STDs.

[0085] QC samples were prepared using commercial crop extracts enriched with unlabeled peptides at three different concentrations (low, medium, and high). Tables 5 and 6 show the nominal concentrations.

[0086] [Table 5]

[0087] Concentration (nominal) of standard substances in commercial crops

[0088]

[0089] a DW - Dry weight

[0090] [Table 6]

[0091] Concentration of QC samples in commercial crops (nominal a )

[0092]

[0093] a Nominal = Nominal concentration before adjustment to QC0 (endogenous level of unlabeled peptides)

[0094] synthetic peptide

[0095] The purified and quantified SIL and unlabeled synthetic peptides used in this study are listed in Table 7. The synthetic peptides were supplied by JPT Peptide Technologies GmbH and stored at a nominal temperature of -20°C until use.

[0096] [Table 7]

[0097] List of synthetic peptides

[0098]

[0099] Analysis method

[0100] The following experimental and data evaluation steps were involved in the validation process for an LC-MS / MS-based method for determining the quantification of HPPD proteins in commercial crops. Briefly, the LC-MS / MS method for identifying and quantifying surrogate peptides of HPPD in a complex biological matrix comprises: (i) weighing freeze-dried commercial crops; (ii) homogenizing / extracting proteins from plant species / matrix samples in lysis buffer; (iii) centrifuging the samples to separate soluble and insoluble proteins to enrich the proteins of interest and reduce sample complexity; (iv) digesting the soluble protein samples with trypsin; (v) centrifuging the samples; (vi) adding SIL peptides (fixed or variable depending on evaluation); (vii) desalting by solid-phase extraction using cation exchange to minimize matrix effects or interference and reduce ion inhibition; and (viii) analyzing the samples by LC-MS / MS. The surrogate peptides of interest unique to the HPPD protein and the corresponding SIL peptides for each commercial crop were measured by LC-MS / MS. MultiQuant TM Data was analyzed using software (version 3.0.2), whereby the chromatographic peak areas of each surrogate peptide (unlabeled) and each corresponding SIL peptide were combined for each sample.

[0101] For the resonance curve, the peak area of ​​the SIL peptide was determined for each surrogate peptide. Subsequently, for each surrogate peptide, the peak area of ​​the SIL peptide for each standard sample was plotted on the y-axis as a function of protein concentration (x-axis) to generate a resonance curve.

[0102] For the QC samples, the peak area ratio (peak area of ​​unlabeled peptide / peak area of ​​the corresponding SIL peptide) was determined. The concentration of the unlabeled peptide in the QC samples was calculated as follows:

[0103]

[0104] Sample processing

[0105] 10 mg to 18 mg of each commercial crop was weighed and added to tubes containing Matrix A dissolution beads (MP Biomedicals). Dissolution buffer (0.1% RapiGest in phosphate-buffered saline (PBS)). TM (Waters)) then add to each sample tube, and FastPrep ® Homogenization was performed using a -24°C homogenizer (MP Biomedicals). Subsequently, the sample was centrifuged at 4°C to remove insoluble matter. The supernatant was transferred; accordingly, it was pooled and diluted in 0.1% RapiGest in PBS as described in Table 8. This diluted commercial crop was enriched with unlabeled peptides to produce QC samples of various concentrations or with SIL peptides to produce standards.

[0106] [Table 8]

[0107] Matrix dilution factor

[0108]

[0109] For each processed standard and QC sample, proteins were denatured by adding an equal volume of 2,2,2-trifluoroethanol (TFE), followed by digestion using 0.1 μg / μL trypsin while incubating the TFE to 10% with 100 mM ammonium bicarbonate for 14 to 18 hours at 37°C. After digestion, the samples were acidified to a final formic acid (FA) concentration of 5%, and SIL peptides were added to each sample at variable or fixed concentrations according to validation evaluation, excluding blanks and carryover blanks. Subsequently, the samples were desalted by solid-phase extraction using mixed-mode cation exchange (MCX) μ-elution plates. The eluent was collected, transferred to two MS plates, evaporated to a dry state, and stored at -20°C (nominal) until MS analysis.

[0110] The processed sample was resolubilized in 11 μL of 92.5 / 7.5 water / acetonitrile (ACN) + 0.2% FA, followed by sonication, vortexing, and centrifugation. NanoAcquity Ultra-High Performance Liquid Chromatography (UPLC) coupled to a QTRAP 6500 mass spectrometer (AB Sciex) ® Eight microliters of material per sample were injected into the (Waters) phase. Peptide separation was achieved using a HALO peptide ES-C18 50 mm x 0.5 mm, 2.7 μm column (Canadian Life Science). The LC gradients used are shown in Table 9 below. The flow rate was 28,000 μL / min. The analytes were measured in cationic mode using a Turbo V MS source. Analyst ® Data acquisition was performed using version 1.6 (AB Sciex).

[0111] [Table 9]

[0112] LC gradient of LC-MS / MS calibration

[0113]

[0114] a DMSO - Dimethyl sulfoxide

[0115] The surrogate peptide and the corresponding SIL peptide are unique to the HPPD protein. The chromatographic peak areas of each surrogate peptide (unlabeled) and each corresponding SIL peptide were aggregated for each sample using MultiQuant version 3.0.2 (AB Sciex).

[0116] For the resonance curve, the peak area of ​​the SIL peptide was determined for each surrogate peptide. A resonance curve was generated by plotting the peak area of ​​the SIL peptide for each standard sample on the y-axis as a function of protein concentration (x-axis).

[0117] For the QC sample, the peak area ratio (peak area of ​​unlabeled peptide / peak area of ​​corresponding SIL peptide) was determined. The concentration of the unlabeled peptide in the QC sample was calculated as described above.

[0118] Microsoft Office Excel ® The mean, standard deviation (SD), and coefficient of variation (CV) were calculated using software. Unrounded values ​​were used for the calculation of the mean, SD, and CV, and were subsequently rounded appropriately in the schematic table.

[0119] Specificity

[0120] Specificity was determined by the ability of the method to measure and distinguish surrogate peptides in the presence of various plant matrix components in the samples. Specificity was measured by determining the presence or absence of each unique surrogate peptide for HPPD proteins by LC-MS / MS.

[0121] As described below, for all commercial crop extracts, assay specificity was evaluated using quantifier and qualitative transfer for unlabeled peptides, and using only quantifier transfer for SIL peptides.

[0122] Unlabeled peptide

[0123] The following analysis was performed:

[0124] Test specificity was evaluated by comparing the mean ratio of the two transitions (quantitative / qualitative) monitored in degraded bovine serum albumin (BSA) buffer (n=3) enriched with unlabeled peptides against the blank (n=3). The blank is considered to be a matrix containing detectable amounts of endogenous HPPD protein processed without the addition of SIL or unlabeled peptides.

[0125] The difference % was calculated using the following formula:

[0126]

[0127] The suitability of the specificity assessment for unlabeled peptides was confirmed using the following acceptance criteria: the difference % between the blank and the buffer must be within 30.0%.

[0128] SIL peptide

[0129] The following analysis was performed:

[0130] Test specificity was evaluated by determining the peak area of ​​the SIL peptide (quantifier transfer) in QC0 (endogenous, n=3) relative to the blank (n=3). The blank is considered to be a matrix containing a detectable amount of endogenous HPPD protein processed without the addition of SIL or unlabeled peptides.

[0131] The percentage of QC0 (endogenous) was calculated using the following formula:

[0132]

[0133] The suitability of the specificity evaluation for SIL peptides was confirmed using the following acceptance criteria: the average SIL peptide peak area in the blank sample must be 5.0% or less of the average peak area of ​​the SIL peptide in the QC0 (endogenous) sample.

[0134] Linearity

[0135] Linearity is the ability of a method to derive results mathematically defined by the amount of analyte in the sample and the response. Linearity was evaluated based on the accuracy of the method. For HPPD in each plant species / matrix, the simplest regression model defining a reflection curve—namely, the linear curve fit—was used. The model was applied based on the quality of the fit using correlation coefficients (R-values).

[0136] Data weights of 1 / x were applied to all peptides. The same regression model and the same data weights were applied to all test runs for each plant species / matrix.

[0137] The suitability of the linearity evaluation was verified using the following continuation acceptance criteria: (1) at least 75.0% of the non-zero standards must be valid, and there must be no deviation exceeding 20.0% of the nominal concentration, except for the LLOQ, where the deviation must not exceed 25.0%; and (2) the interpolation curve must have R ≥ 0.9900.

[0138] Quantitative limit

[0139] LLOQ and ULOQ are the minimum and maximum concentrations at which the response of the surrogate peptide can be determined within acceptable limits of accuracy. LLOQ and ULOQ were determined for the surrogate peptide in each plant species / matrix using the reverberation curves as described.

[0140] The conformity of the LLOQ and ULOQ assessments was verified using the following acceptance criteria: LLOQ and ULOQ standards must meet the accuracy criteria specified above.

[0141] The quantitative range for HPPD is the interval between the upper and lower concentration limits where the analytical procedure is proven to have an appropriate level of accuracy and linearity (see Table 5 for standard concentrations). Using data obtained from the linearity evaluation of this method, the quantitative range for HPPD in each plant species / matrix was determined. The high and low concentrations tested as the quantitative range for the procedure were effectively validated by determining accuracy.

[0142] Carryover is the presence of the analyte in a subsequent injection. In this study, carryover was determined during the linearity evaluation by evaluating two blank samples injected after each ULOQ standard. There should be no interference between them. Carryover was evaluated for all linearity progressions by applying the following progression acceptance criteria. The first carryover blank sample injected after the ULOQ standard was evaluated. The second carryover blank was evaluated but did not meet the acceptance criteria. At least 50% of the first carryover blank sample injected after the ULOQ standard must be within acceptable interference at the following retention times, as described below: SIL peptide: peak area must be 20.0% or less of the average peak area of ​​the SIL peptide of the LLOQ standard(s). Additionally, for the stability evaluation as well as for precision and accuracy, carryover after injection of the high QC sample was evaluated by analyzing the resolubility buffer (RSB) sample. The order of carryover evaluation was as follows: high QC, followed by two RSB samples.

[0143] Criteria for allowing progress

[0144] As part of the verification, QC samples were included to demonstrate the acceptability of the progress, as well as the stability and accuracy. For the aforementioned progress to be considered valid, the following criteria must be met. Each plant species / matrix was treated independently against the acceptance criteria.

[0145] QC Sample

[0146] QC samples provide criteria for allowing or rejecting progress. QC samples were prepared by reinforcing the sample matrix with unlabeled peptides at known concentrations. The suitability of QC samples using low QC (QC1), medium range QC (QC2), and high QC (QC3) was verified using the following acceptance criteria: (1) at least 50% of the QC samples at each low, medium, and high concentration must be within ±20.0% deviation; and (2) at least 67% of the QC samples across the low, medium, and high concentrations must be within ±20.0% deviation of their nominal values.

[0147] Precision and accuracy

[0148] Precision is the degree of agreement between individual test results when the procedure is repeatedly applied to multiple sampling of homogeneous samples. Accuracy is the degree of agreement between the confirmed value and the allowed reference value when the procedure is repeatedly applied to multiple sampling of homogeneous samples. In this study, the precision and accuracy of HPPD proteins in each plant species / matrix for LC-MS / MS assays were determined by evaluating test-versus-test and analyte-versus-analyte variability.

[0149] Method precision and accuracy were evaluated using QC samples. Precision and accuracy tests were performed using QC samples at three concentrations (low, medium, and high) (Tables 5 and 6). QC samples were prepared from commercial crop extracts and enriched with unlabeled peptides.

[0150] Precision (CV) and accuracy (bias) were evaluated over three independent test runs using two different analytes spread over three days.

[0151] Each precision and accuracy test was performed with three replicas per QC concentration. QC0 (endogenous) was also included and analyzed in triples.

[0152] The bias % was calculated using the following formula:

[0153]

[0154] Precision and accuracy within the test were evaluated using precision and accuracy progressions that met the progress acceptance criteria specified in Section 0. Then, precision and accuracy between the test were evaluated using precision and accuracy progressions that met the within-test criteria. The suitability of the precision and accuracy evaluations for within-test and between-test progressions was verified using the following acceptance criteria: (1) the CV for QC0 samples must be 25.0% or less; and (2) the CV for low, medium, and high concentration QC samples must be 20.0% or less and within ±20.0% bias.

[0155] In addition, the maximum injection time for the test progression for the study sample analysis was established by examining the batch size of the precision and accuracy progressions. The precision and accuracy progression with the maximum batch size was used to determine the maximum injection time for the study sample analysis. Furthermore, a trend analysis of the QC0 (endogenous) samples was performed.

[0156] Extraction efficiency is the amount of protein of interest (i.e., HPPD) recovered from the matrix. Extraction efficiency was determined by successive sequential extractions. If 5.0% or less of the total protein is recovered in the last round, the extraction recovery is considered final. The efficiency of the protein extraction method using commercial crops was evaluated through repeated extraction of HPPD.

[0157] One analyte was extracted from three copies of each commercial crop. Insoluble material was collected and extracted at least three times, and the supernatant from each extraction was retained for analysis.

[0158] The extraction efficiency for each sample was calculated using the following formula:

[0159]

[0160] The suitability of extraction efficiency for each protein was verified using the following acceptance criteria: Sequential extraction will be considered complete when the final extraction result is 5.0% or less of the total material recovered for each individual protein from all combined extractions:

[0161]

[0162] The extraction efficiency (average of copies of the first iteration) is expected to be 60.0% or higher, and the precision must be 20.0% or lower for the extraction of the first round.

[0163] stability

[0164] The stability of HPPD proteins in given plant species / matrices under specific conditions during a given time interval was evaluated. For surrogate peptides in processed samples, the stability of HPPD proteins in each plant species / matrix was determined.

[0165] Processed sample stability

[0166] The stability of the processed samples of the dried extract was analyzed at two different concentrations (low and high) with three replicates per concentration. Newly prepared and processed QC samples were analyzed by LC-MS / MS (considered Day 0). The dried stability samples were not analyzed immediately by LC-MS / MS but were stored for a predetermined period.

[0167] Dry stability samples were stored at a nominal temperature of -20℃ for 6 days (146 hours and 16 minutes), then re-solubilized and analyzed by LC-MS / MS.

[0168] The stability of the surrogate peptide in the processed sample was verified using the following acceptance criteria: (1) the overall average peak area ratio of the processed Day 0 and stability samples must have a CV of 25.0% or less; and (2) the percentage difference in the peak area ratio between the stability and Day 0 QC (processed on the same injection day) must be within ± 25.0%.

[0169]

[0170] According to the verification protocol, carryover evaluation is adequately outlined in the overall description, which specifies that peak area is used for carryover evaluation; however, due to an error, the peak area ratio is specified in the description verified in the acceptance criteria. Since peak area is the correct method for performing the evaluation, interference was evaluated using peak area in this study. Therefore, because a suitable approach for evaluating interference in the carryover blank was applied (i.e., peak area), there is no impact on data quality and integrity.

[0171] According to the validation protocol, the peak area ratio (SIL / unlabeled) for each peptide of the quantifier transition is used to plot the calibration curve data on a linear scale. However, the peak area for each peptide of the quantifier transition was used instead for plotting. The peak area ratio cannot be used for linearity assessment due to the influence of unlabeled signals from stable isotope-labeled peptides at high spiking concentrations. Since linearity assessment was performed, there is no impact on data quality and integrity.

[0172] According to the verification protocol, extraction efficiency evaluation is performed using four sequential extractions (rounds). For the two matrices (i.e., barley and wheat seeds), the extraction efficiency results did not meet the acceptance criteria outlined below.

[0173] The result of the barley seed - final extraction (Round 4) was 8.2% (acceptable standard: <5.0%).

[0174] Wheat seeds - The result of the final extraction (Round 4) was 6.2% (acceptable criterion: <5.0%). The precision of the extraction in Round 1 was 27.5% CV (acceptable criterion: ≤ 20.0%).

[0175] However, there is no impact on the results of the study for the following reasons: (1) The final extraction results for barley and wheat seeds show that the extraction of HPPD protein was nearly completed after four extraction rounds. Additionally, in the sample analysis, HPPD concentration was determined from a single extraction (Round 1) and adjusted for extraction efficiency %; (2) In the case of wheat seeds, although the precision of the extraction in Round 1 (27.5% CV) exceeded the 20.0% CV standard, the precision and accuracy results for QC0 (endurance) from three verification runs (Runs 4 to 6) obtained from a single extraction demonstrate that a precision of 20.0% or less was achieved.

[0176] According to the verification protocol, for each precision and accuracy progress, two (2) RSB samples are injected after the high QC sample at the end of each matrix. However, for one matrix (i.e., wheat seeds), the RSB sample injected after the high QC sample (QC3 replica 3) was obtained using the MRM method for peptide GNFSQLFK (SEQ ID NO:2) instead of peptide GNFSELFK (SEQ ID NO:1) due to an error. Therefore, RSB carryover evaluation was not performed for wheat seeds.

[0177] However, the results of the study were not affected for the following reasons: (1) no carryover of peptide GNFSELFK (SEQ ID NO:1) was observed for any other matrix (including wheat feed), and thus the overall conclusion is that peptide GNFSELFK (SEQ ID NO:1) did not show any tendency for carryover, and carryover of peptide GNFSELFK for wheat seed matrix can be inferred to be very unlikely; (2) consequently, based on the available dataset, an acceptable RSB carryover criterion (i.e., 5.0% or less of the non-labeled peptide peak area relative to the QC0 peak area at RT) can be established for sample analysis of all matrices (including wheat seed) in relation to peptide GNFSELFK (SEQ ID NO:1) as well as GNFSQLFK (SEQ ID NO:2).

[0178] In addition, there were minor deviations from the SOP that had no impact on the study. These are recorded in the study file. No other situations that would adversely affect the quality or integrity of the generated data occurred during the conduct of this study.

[0179] Specificity

[0180] The specificity of the assay was evaluated for unlabeled and SIL peptides in all commercial crop extracts. The evaluation revealed some minor interference in the retention times of surrogate peptides and corresponding SIL peptides in the tested commercial crop samples. For unlabeled peptides, the percentage difference in the average ratio of the two transitions between the blank and buffered samples was less than 30.0% for all tested plant species / matrixes, and since all interference was less than 5.0% of the average peak area of ​​the SIL peptide in the QC0 (endogenous) sample, the acceptance criteria for specificity were met.

[0181] Linearity

[0182] Linearity for HPPD proteins in each plant species / matrix was determined using reciprocal curves as described in Sections 3.4 and 0. The simplest regression model used to define reciprocal curves, namely linear regression, was utilized. Data weights of 1 / x were applied to all plant species / matrices. Table 10 summarizes the standard curve parameters (slope, intercept, and R-value) for the linearity assessment. The correlation coefficient (R) of the standard curve for HPPD proteins in each plant species / matrix was greater than or equal to 0.9900. All established data fell within the acceptance criteria as described in Section 0 and were confirmed to be suitable for linearity assessment.

[0183] [Table 10]

[0184] Reflection curve parameters in commercial crops for a linear equation with a weighting factor of 1 / x

[0185]

[0186] Standard concentrations of the inverse curves calculated from each plant species / matrix are provided in Table 11.

[0187] [Table 11]

[0188] Reverse curve standard concentrations from commercial crops

[0189]

[0190] For each standard of each plant species / matrix, N=2

[0191] Quantitative limit

[0192] In the linearity assessment, the accuracy (bias) was within ±25.0% and ±20.0% for the LLOQ and ULOQ samples, respectively (Table 11), and thus the quantification limits were set at the lowest and highest non-zero standard concentrations. Table 12 summarizes the upper and lower quantification limits for HPPD for each plant species / matrix.

[0193] [Table 12]

[0194] Quantification limit for HPPD in commercial crops

[0195]

[0196] The quantitative range for HPPD in each plant species / matrix is ​​the interval between the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) concentrations at which the analytical procedure was proven to have an appropriate level of accuracy. LLOQ and ULOQ samples met the accuracy criteria for HPPD in each plant species / matrix. The quantitative range of the method was established for each plant species / matrix (Table 13).

[0197] [Table 13]

[0198] Quantitative range of HPPD in commercial crops

[0199]

[0200] Carryover

[0201] Carryover was determined in the linearity evaluation by evaluating blank samples injected after the ULOQ standard for the tested commercial crop samples. The evaluation did not show interference with the retention time of the SIL peptide. The carryover acceptance criteria were met because the SIL peptide peak area ratio was 20.0% or less of the average peak area ratio of the SIL peptide of the LLOQ standard(s) in at least 50% of the first carryover blank samples injected after the ULOQ standard.

[0202] In addition, precision and accuracy, as well as carryover during stability progression, were evaluated using RSB samples following injection of high QC samples. Overall, the evaluation showed no carryover in the blank samples. The acceptance criterion for RSB blank carryover in the analysis of the study samples would be 5.0% or less of the average peak area of ​​the unlabeled peptide in the QC0 (endogenous) samples.

[0203] Precision and accuracy

[0204] The precision and accuracy of the HPPD method for commercial crops were evaluated using the QC samples described herein. Three precision and accuracy trials were conducted. All met the criteria for accepting trials. Precision (CV) and accuracy (bias) data for intra-test and inter-test trials are summarized in Tables 14 through 23 (one table per plant species / matrix).

[0205] For both within-test and between-test cases, the CV was less than 25.0% for the QC0 sample and less than 20.0% for the low, medium, and high QC samples, so the method precision evaluation was appropriate.

[0206] For both within-test and between-test cases, the bias was within ±20.0% for low, medium, and high QC samples; therefore, the evaluation of method accuracy was appropriate.

[0207] The longest injection time for the precision and accuracy batch was approximately 36 hours and 58 minutes (batch size of 187 injections), and thus this was established as the longest injection time for the assay for the analysis of the study sample.

[0208] Trend analysis of QC0 (endogenous) samples was performed from the progression of precision and accuracy. In addition, to extend the trend analysis, QC0 (endogenous) samples were analyzed for sample analysis.

[0209] [Table 14]

[0210] Results of intra-test and inter-test precision and accuracy for HPPD in barley seeds

[0211]

[0212] Nominal = Actual concentration used in calculation

[0213] N=3 for each QC in each process

[0214] N / Ap = Not applicable

[0215] [Table 15]

[0216] Results of intra- and inter-test precision and accuracy for HPPD in Maze seeds

[0217]

[0218] Nominal = Actual concentration used in calculation

[0219] N=3 for each QC in each process

[0220] N / Ap = Not applicable

[0221] [Table 16]

[0222] Results of intra-test and inter-test precision and accuracy for HPPD in rice seeds

[0223]

[0224] Nominal = Actual concentration used in calculation

[0225] N=3 for each QC in each process

[0226] N / Ap = Not applicable

[0227] [Table 17]

[0228] Results of intra-test and inter-test precision and accuracy for HPPD in soybean seeds

[0229]

[0230] Nominal = Actual concentration used in calculation

[0231] N=3 for each QC in each process

[0232] N / Ap = Not applicable

[0233] [Table 18]

[0234] Results of intra-test and inter-test precision and accuracy for HPPD in wheat seeds

[0235]

[0236] Nominal = Actual concentration used in calculation

[0237] N=3 for each QC in each process

[0238] N / Ap = Not applicable

[0239] [Table 19]

[0240] Results of intra- and inter-test precision and accuracy for HPPD in barley feed

[0241]

[0242] Nominal = Actual concentration used in calculation

[0243] N=3 for each QC in each process

[0244] N / Ap = Not applicable

[0245] [Table 20]

[0246] Results of intra- and inter-test precision and accuracy for HPPD in Maze Feed

[0247]

[0248] Nominal = Actual concentration used in calculation

[0249] N=3 for each QC in each process

[0250] N / Ap = Not applicable

[0251] [Table 21]

[0252] Results of intra- and inter-test precision and accuracy for HPPD in oat feed

[0253]

[0254] Nominal = Actual concentration used in calculation

[0255] N=3 for each QC in each process

[0256] N / Ap = Not applicable

[0257] [Table 22]

[0258] Results of intra- and inter-test precision and accuracy for HPPD in soybean feed

[0259]

[0260] Nominal = Actual concentration used in calculation

[0261] N=3 for each QC in each process

[0262] N / Ap = Not applicable

[0263] [Table 23]

[0264] Results of intra- and inter-test precision and accuracy for HPPD in wheat feed

[0265]

[0266] Nominal = Actual concentration used in calculation

[0267] N=3 for each QC in each process

[0268] N / Ap = Not applicable

[0269] Extraction efficiency

[0270] Table 24 summarizes the efficiency of the protein extraction method from commercial crops. For HPPD, all plant species / matrixes reached acceptable extraction efficiency in a single replicate. The average extraction efficiency of HPPD from barley seeds (59.9%), maize seeds (69.8%), rice seeds (73.9%), soybean seeds (64.9%), wheat seeds (54.3%), barley feed (69.8%), maize feed (76.3%), oat feed (78.4%), soybean feed (65.4%), and wheat feed (77.2%) demonstrates that the method meets the evaluation of acceptable extraction efficiency. Furthermore, the CV value for HPPD was less than 20.0% for all plant species / matrixes, and the amount at final extraction was less than 5.0% for all commercial crops, except for barley and wheat seeds.

[0271] [Table 24]

[0272] Extraction efficiency of HPPD from commercial crops

[0273]

[0274] N = 4 extraction iterations and 3 copies per iteration

[0275] Extraction efficiency was performed in Process 3.

[0276] For details, refer to Table A14 in Appendix A.

[0277] Values ​​in italics and bold are outside the acceptable criteria; refer to Section 5.1.3.

[0278] Table 25 summarizes the stability of the dry extract of the surrogate peptide for 6 days (146 hours and 16 minutes) at a nominal temperature of -20℃. The average peak reaction rate of the stability samples was a CV of 25.0% or less, and the difference % of the stability samples was within ±25.0% for the stability evaluation of the dry processed samples compared to the Day 0 QC samples.

[0279] [Table 25]

[0280] Stability of commercial crop dry extracts for 6 days (146 hours 16 minutes) at a nominal temperature of -20℃

[0281]

[0282] N = 3 for all QCs; N / Ap = Not applicable

[0283] a Difference % - (Stability QC - Day 0 QC) / Day 0 QC x 100

[0284] Drying stability was performed in process 7.

[0285] For details, refer to Table A15 in Appendix A.

[0286] The purpose of this study is to investigate various commercial crops (barley, maize, rice, soybean, and wheat seeds, and barley, maize, oat, soybean, and wheat feed) using LC-MS / MS. p- The goal was to validate an MS-based method for the quantification of hydroxyphenylpyruvate deoxygenase (HPPD) protein. Relative concentrations were determined using surrogate peptides unique to HPPD protein. Using commercial crop samples, method performance parameters including specificity, linearity, limit of quantification, carryover, precision and accuracy, extraction efficiency, and stability were evaluated.

[0287] Specificity evaluation did not show significant interference with the retention times of unlabeled peptides (mean ratio difference between the two transitions between blank and buffer was less than 30.0%) and SIL peptides (SIL signal of QC0 (endogenous) was less than 5.0%) in any commercial crop sample tested. All specificity parameters met the acceptance criteria.

[0288] The lower limit and quantification range (fmol / mg DW and μg / g DW) for HPPD in commercial crops are shown in Table 26.

[0289] [Table 26]

[0290] List of commercial crops, surrogate peptides, quantification lower limits, and quantification ranges

[0291]

[0292] The precision and accuracy within and between tests of the method are summarized in Table 27.

[0293] [Table 27]

[0294] Range of intra-test and inter-test precision and accuracy for HPPD in commercial crops

[0295]

[0296] A linear equation was determined to appropriately represent the concentration / detector response relationship for HPPD in all commercial crops. Data weights of 1 / x were used for all commercial crops.

[0297] The efficiency of the protein extraction method was 59.9% for barley seeds, 69.8% for maize seeds, 73.9% for rice seeds, 64.9% for soybean seeds, 54.3% for wheat seeds, 69.8% for barley feed, 76.3% for maize feed, 78.4% for oat feed, 65.4% for soybean feed, and 77.2% for wheat feed. The CV of the first round of extraction was less than 20.0% for all commercial crops, except for wheat seeds, which had a CV of 27.5%. However, the precision and accuracy results of QC0 (endurance) for wheat seeds obtained from a single extraction demonstrate that a precision of less than 20.0% was achieved.

[0298] Processing stability (dried extract) was evaluated at -20°C for 6 days. The stability evaluation met the acceptance criteria. CV was 25.0% or less, and the % difference in peak area ratio between stability and Day 0 QC was within ± 25.0% for all commercial crops.

[0299] All evaluated performance parameters met the prescribed acceptance criteria, with the exception of the final extraction percentages for barley and wheat seeds, which were 8.2% and 6.2%, respectively, as well as the precision of the first round extraction for wheat seeds, which was 27.5% CV. In the sample analysis, the concentration of HPPD is determined from a single extraction (first round) and adjusted for extraction efficiency %; since precision of 20.0% or less was achieved for wheat seeds in the precision and accuracy progression, there is no impact on the study results. Based on the results of this study, mass spectrometry-based methods were confirmed to be suitable for the quantification of HPPD proteins in the list of commercial crops evaluated in this study according to GLPS.

[0300] Although the present invention has been described in connection with specific embodiments thereof, it will be understood that further modifications of the device of the present invention are possible. This patent application is intended to include any variation, use, or modification of the present invention (generally following the principles of the present invention, falling within the known or customary practices within the art to which the invention pertains, and applicable to the essential features of the present invention prior to description, and including deviations from the present disclosure within the scope of the appended claims).

[0301] All publications and patent applications mentioned herein represent the level of skill of those skilled in the art to which the present invention pertains. All publications and patent applications herein are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated by reference.

Claims

Claim 1 Among a mixture of proteins from one or more biological samples from one or more crops p- A labeled surrogate peptide that functions in a mass spectroscopic assay to selectively detect or quantify hydroxyphenylpyruvate deoxygenase (HPPD) protein, wherein the labeled surrogate peptide comprises an amino acid sequence consisting of an isotope label and GNFSELFK (SEQ ID NO:1) or GNFSQLFK (SEQ ID NO:2). Claim 2 In claim 1, the labeled surrogate peptide is a labeled surrogate peptide labeled by incorporating a stable isotope-labeled (SIL) amino acid. Claim 3 In paragraph 2, the SIL amino acid is lysine, a labeled proxy peptide. Claim 4 In paragraph 1, the label surrogate peptide, wherein the crop is barley, rice, soybean, wheat, oats, or maize. Claim 5 In paragraph 4, the crop is barley, rice, soybean, or wheat, and the labeled surrogate peptide is a labeled surrogate peptide comprising an isotope label and the amino acid sequence of SEQ ID NO:

1. Claim 6 In paragraph 4, the crop is a maize, and the labeled surrogate peptide is a labeled surrogate peptide comprising an isotopic label and the amino acid sequence of SEQ ID NO:

2. Claim 7 Assay cassette comprising at least two labeled surrogate peptides having amino acid sequences selected from the group consisting of GNFSELFK (SEQ ID NO:1) and GNFSQLFK (SEQ ID NO:2). Claim 8 Target p- A method for simultaneously detecting or quantifying one or more target HPPD proteins in a complex biological sample from a crop containing a mixture of hydroxyphenylpyruvate deoxygenase (HPPD) proteins and non-target proteins, comprising: a. obtaining a biological sample from the crop; b. extracting proteins from the biological sample to produce an extract containing a mixture of proteins; c. reducing the amount of insoluble proteins in the extract of step b to produce an extract of concentrated soluble proteins; d. degrading the soluble proteins in the extract of step c to produce an extract containing peptide fragments, wherein the peptide fragments contain unlabeled surrogate peptides specific to a given target HPPD protein; e. concentrating the peptide fragments in the extract of step d; f. A step of adding one or more synthetic labeled surrogate peptides comprising isotope labeling and the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 to the extract of step e to produce a peptide fragment mixture, wherein each synthetic labeled surrogate peptide in the peptide fragment mixture is an analog of an unlabeled surrogate peptide specific to a given target HPPD protein and has the same amino acid sequence as the unlabeled surrogate peptide of the given target HPPD protein, and different synthetic labeled surrogate peptides are added for each target HPPD protein such that the number of added labeled surrogate peptides is equal to the number of target HPPD proteins in the peptide fragment mixture; g. a step of concentrating the unlabeled surrogate peptide and the labeled surrogate peptide by reducing the amount of the unlabeled surrogate peptide in the mixture; h. a step of dissolving the peptide fragment mixture from step g through liquid chromatography; i.A method comprising: a step of analyzing a mixture of peptide fragments generated from step h through mass spectrometry, wherein the detection of a transfer ion fragment of an unlabeled surrogate peptide and a transfer ion fragment of a synthetic labeled surrogate peptide indicates the presence of a target HPPD protein specific to said labeled surrogate peptide; and j. a step of calculating the amount of target HPPD protein in a biological sample by comparing a mass spectrometry signal generated from a transfer ion fragment of an unlabeled surrogate peptide with a mass spectrometry signal generated by a transfer ion fragment of a synthetic labeled surrogate peptide. Claim 9 In claim 8, the crop is barley, rice, soybean, or wheat, and the synthetic labeled surrogate peptide comprises an isotope label and the amino acid sequence of SEQ ID NO:

1. Claim 10 In claim 8, the crop is maize, and the synthetic label surrogate peptide comprises an isotopic label and the amino acid sequence of SEQ ID NO:

2. Claim 11 A method according to claim 9 or 10, wherein the synthetic labeled surrogate peptide is labeled by incorporating a stable isotope-labeled (SIL) amino acid. Claim 12 In paragraph 11, the SIL amino acid is lysine, method.

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