Compositions and Methods for Protein Detection

Abstract

The present invention generally relates to peptide biomarkers having specific ionization characteristics for directly quantifying one or more target HPPD proteins in biological samples, including crop plant samples, by liquid chromatography tandem mass spectrometry multiple reaction monitoring (MRM). The peptide biomarkers, in combination with an MRM-based method, can be used to quantify a single target protein or multiple target proteins within a crop plant, such as maize, either alone or in combination using selected peptide biomarkers. The present disclosure allows for broad-based, reliable quantification within different biological matrices, including plant matrices. Also provided are different combinations of peptide biomarkers that can be used to implement the methods of the present invention.

Description

[0001] Reference to the electronically submitted Sequence Listing

[0002] An official copy of the Sequence Listing was electronically submitted via EFS-Web as a Sequence Listing in ASCII format in a file named "81875-WO-REG-ORG-P-1_SeqList.txt" generated on April 20, 2020, and the Sequence Listing is 1 kilobyte in size and was submitted contemporaneously with this specification. The Sequence Listing contained in this ASCII format file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention generally relates to the use of mass spectrometry for the selective detection, quantification, and characterization of target proteins in complex biological samples. Background Art

[0004] Immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)) are currently the preferred method in the agricultural industry for detecting and quantifying plant endogenous proteins or proteins introduced through plant genetic modification. A key part of an immunoassay is an antibody specific to the target protein (antigen). Immunoassays can be highly specific, and samples typically only require simple preparation before being analyzed. In addition, immunoassays can be used qualitatively or quantitatively over a wide concentration range. Typically, immunoassays require a separate test for each target protein. Antibodies can be polyclonal, produced in animals, or monoclonal, produced by cell cultures. By their nature, a mixture of polyclonal antibodies will have multiple recognition epitopes, which can increase sensitivity but may also reduce specificity because the likelihood of sequence and structural homology with other proteins increases with the number of different antibody complementarity-determining regions present. Monoclonal antibodies have some advantages over polyclonal antibodies because they exhibit uniform affinity and specificity for a single epitope or antigenic determinant and can be produced in large quantities. However, there are inherent properties in all antibodies that limit their use in more demanding applications, such as the selective detection and quantification of individual proteins in complex mixtures of similar transgenic or endogenous proteins. In addition, both polyclonal and monoclonal antibodies may require further purification steps to enhance sensitivity and reduce background in the assay. In addition, ELISA systems may not be able to detect subtle changes in the target protein that can have a significant impact on its physical and biological properties. For example, the antibody may not recognize a specific form of a protein or peptide that has been altered by post-translational modifications (such as phosphorylation or glycosylation), has a blurred conformation, or has been partially degraded. Identifying such modifications is crucial because changes in the physical and biological properties of these proteins can play an important role in their enzymatic, clinical, or other biological activities. Such changes may limit the reliability and utility of ELISA-based quantification methods.

[0005] Currently, the effective identification and / or quantification of proteins in crop plants depends on the accuracy of immunoassays. The development of successful immunoassays depends on certain characteristics of the antigen used to develop the antibody, namely the size, hydrophobicity, and tertiary structure of the antigen, as well as the quality and accuracy of the antibody. The specificity of the antibody must be carefully examined to elucidate any cross-reactivity with similar substances, which may lead to false positive results. The problem in the industry currently is that many antibodies in commercially available test kits cannot distinguish between similar proteins in various proteins of different crop plants.

[0006] 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 seen important advances in targeted protein analysis, such as multiple reaction monitoring (MRM) by liquid chromatography-tandem mass spectrometry (LC-MS / MS) with electrospray ionization. The basic concept is that proteins can be quantified by measuring their specific constituent peptides (surrogate peptides) after proteolytic digestion. Data acquisition only for the selected peptides allows measurements with higher precision, sensitivity, and throughput. Protein quantification by MRM-based measurement of surrogate peptides is the fastest growing application of MS in protein analysis. Compared to immuno-based assays, MRM-based protein assays have two compelling advantages. The first advantage is the ability to systematically configure specific assays for essentially any protein without the use of antibodies. The second advantage is the ability of targeted MS assays to multiplex many peptides in a single analysis. In addition, MRM is a direct analysis, while immuno-based assays are indirect analyses. Immuno-based assays rely on binding assays that include a linking reagent that can be immobilized on a solid phase and a detection reagent that will specifically bind and use an enzyme to generate a signal that can be correctly quantified.

[0007] Accordingly, there is a continuing need to identify surrogate peptides that have all of the biochemical properties required to function in MRM-based assays and have additional properties such that these surrogate peptide pairs have absolute specificity for target proteins that may have mostly overlapping amino acid sequences, i.e., one or more transitions of the surrogate peptides are able to clearly and without interference distinguish between two closely related target proteins in multiple complex matrices. Such selective surrogate peptides and their transitions should be able to distinguish between target proteins that are similar to each other or similar to transgenic proteins in transgenic crop plants. SUMMARY OF THE INVENTION

[0008] The present invention provides labeled surrogate peptides and their respective transition ions that can be used to selectively detect or quantify target proteins in complex biological matrices using mass spectrometry. The present invention further provides methods and systems for selectively detecting or quantifying target HPPD proteins in complex biological matrices using the labeled surrogate peptides and transition ions.

[0009] In one aspect of the present invention, an internal standard peptide label is designed through empirical analysis and computer simulation digestion analysis; and is chemically synthesized with heavy amino acid residues or genetically synthesized by expressing a synthetic gene in the presence of one or more amino acids or metabolic intermediates labeled with stable isotopes. In certain embodiments, the internal standard can be individually characterized by mass spectrometry (MS) analysis, including tandem mass spectrometry (MS / MS), and more specifically, liquid chromatography-tandem mass spectrometry (LC-MS / MS). After characterization, preselected parameters of the peptide can be collected, such as the monoisotopic mass of each peptide, its alternative charge states, alternative m / z values, m / z transition ions, and the ion type of each transition ion. Other considerations include optimizing peptide size, avoiding post-translational modifications, avoiding process-induced modifications, and avoiding a high rate of protease cleavage omissions.

[0010] In one aspect, the present invention provides a labeled alternative peptide that functions in mass spectrometry analysis to selectively detect or quantify 4-hydroxyphenylpyruvate dioxygenase (HPPD) protein in a protein mixture in one or more biological samples from one or more crop plants, the alternative peptide comprising 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 alternative peptide is labeled by incorporating a stable isotope-labeled (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, oats, or maize. In additional embodiments, the crop plant is barley, rice, soybean, wheat, or rice, and the alternative peptide comprises a label and the amino acid sequence of SEQ ID NO:1. In additional embodiments, the crop is maize, and the alternative peptide comprises a label and the amino acid sequence of SEQ ID NO:2.

[0011] In one aspect, the present invention provides a test kit comprising at least two labeled alternative peptides as described in the present invention.

[0012] In one aspect, the present invention provides a method for simultaneously detecting or quantifying one or more target HPPD proteins in a complex biological sample, the complex biological sample being from a crop plant comprising a mixture of the target protein and non-target proteins, the method comprising: (a) obtaining a biological sample from the crop plant; (b) extracting proteins from the biological sample to obtain an extract comprising a protein mixture; (c) reducing the amount of insoluble proteins in the extract of step b to obtain a concentrated extract of soluble proteins; (d) digesting the soluble proteins in the extract of step c to obtain an extract comprising peptide fragments, wherein the peptide fragments comprise at least one alternative peptide specific for the target protein; (e) concentrating the peptide fragments in the extract of step d; (f) adding one or more labeled alternative peptides of the present invention, wherein each labeled alternative peptide has the same amino acid sequence as each alternative peptide of the target protein, and wherein the number of labeled alternative peptides added is equal to the number of target proteins in the mixture; (g) concentrating the alternative peptides and the labeled alternative peptides by reducing the amount of non-alternative peptides in the mixture; (h) resolving the mixture of peptide fragments from step g by liquid chromatography; (i) analyzing the mixture of peptide fragments obtained from step h by mass spectrometry, wherein detection of the transition ion fragments of the labeled alternative peptide indicates the presence of the target protein from which the alternative peptide is derived; and, optionally, (j) calculating the amount of the target protein in the biological sample by comparing the mass spectrometry signal generated by the transition ion fragments of step i with the mass spectrometry signal generated by the transition. In some embodiments, the crop plant is barley, rice, soybean, wheat or rice, and the alternative peptide comprises a label and the amino acid sequence of SEQ ID NO:1. In other embodiments, the crop plant is maize, and the alternative peptide comprises a label and the amino acid sequence of SEQ ID NO:2. In other embodiments, the peptide is labeled by incorporation of stable isotope labeled (SIL) amino acids. In additional embodiments, the SIL amino acids are lysine, isoleucine, valine or arginine.

[0013] Many different combinations of alternative peptides can be monitored and quantified simultaneously by MRM assays with one or more specific alternative peptides from the HPPD proteins of the present invention, and thus provide a method for obtaining the total amount of each of those proteins in a given protein preparation from a biological sample by mass spectrometry measurement. The combination of these peptides with MRM-based assays has many applications, including quantitative peptide / protein analysis for determining expression levels at different growth stages of crop plants, determining expression levels in different crop plant tissues and organs (including but not limited to leaf tissue, seeds and grains, pollen and root tissue), determining potential exposure levels for regulatory risk assessment, determining different levels of proteins in food processing, comparative and generational studies. Broadly speaking, the unique alternative peptides of these seven proteins can be used in combination with MRM assays for a variety of applications, including agricultural applications, bioequivalence testing, biomarker, diagnostic, discovery, food, environmental, therapeutic monitoring in all types of biological and abiotic matrices. In some aspects of the present invention, a kit is provided that comprises one or more labeled alternative peptides of the present invention and permits simultaneous and selective detection or quantification of any one or more target proteins of the present invention.

[0014] The present invention also provides a method for selectively detecting or quantifying a target HPPD protein in a complex biological matrix, such as a biological sample from a crop plant. Such a method includes: obtaining a sample from the crop plant, such as from leaves, seeds or grains, pollen or roots; extracting proteins from the plant sample; concentrating the library of target proteins by reducing the amount of insoluble proteins in the extract; digesting the soluble proteins in the extract with a selected enzyme, such as trypsin, to obtain an extract containing peptide fragments, wherein the peptide fragments contain at least one surrogate peptide specific for each target protein; adding an assay kit for specifically detecting SIL peptides of the target protein, wherein each labeled surrogate peptide has the same amino acid sequence as each surrogate peptide of the target protein, and wherein the number of labeled surrogate peptides added is equal to the number of target proteins in the mixture; concentrating the surrogate peptides and the labeled surrogate peptides by reducing the amount of non-surrogate peptides in the mixture; decomposing the peptide fragment mixture using liquid chromatography; analyzing the peptide fragment mixture using mass spectrometry, wherein detection of the transition ion fragments of the labeled surrogate peptides indicates the presence of the target protein from which the surrogate peptide is derived; and, optionally, calculating the amount of the target protein in the biological sample by comparing the mass spectrometry signal generated by the transition ion fragments with the mass spectrometry signal generated by the transition ion of the labeled surrogate peptide. The SIL surrogate peptides derived from the proteins of the present invention each have unique transition ions in a mass spectrometry-based multiple reaction monitoring (MRM) assay. Thus, these peptides will generate selective MS ions due to minor changes in collision energy, resulting in different degrees of ionization. For example, triple quadrupole MS can be used to generate high m / z ions specific for the peptide. Therefore, the method of the present invention can provide a selectivity advantage over the use of lower m / z strong ion labels known in the art, thereby reducing endogenous background.

[0015] The present invention further provides a system for high-throughput detection or quantification of a target protein. Such a system comprises a kit of pre-designed labeled surrogate peptides specific for the target protein; and one or more mass spectrometers.

[0016] Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the preferred embodiments of the invention, as well as the drawings and sequence listing.

[0017] Brief Description of the Sequences

[0018] SEQ ID NO:1 is the amino acid sequence of a stable isotope-labeled surrogate peptide for selectively detecting and quantifying HPPD proteins in barley, rice, soybeans, wheat and oats.

[0019] SEQ ID NO:2 is the amino acid sequence of a stable isotope-labeled surrogate peptide for selectively detecting and quantifying HPPD proteins in maize. Detailed Description

[0020] This description is not intended to be an exhaustive catalog of all the different ways in which the invention may be practiced or of all the features that may be added to the invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. Additionally, numerous variations and additions to the different embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, without departing from the invention. Accordingly, the following description is intended to illustrate some particular embodiments of the invention and is not an exhaustive recitation of all its permutations, combinations, and variations.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. It should also be understood that the terms used herein are merely for the purpose of describing specific embodiments and are not intended to limit the scope of the invention.General references related to the present invention include: 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, pp. 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, pp. 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.

[0022] Definitions

[0023] As used herein and in the appended claims, the singular forms "a / an" and "the" may mean one or more than one. Thus, for example, reference to "a plant" may refer to a single plant or multiple plants.

[0024] As used herein, "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items, along with the absence of combinations when interpreted in the alternative, "or".

[0025] The term "about" is used herein to mean approximately, roughly, around, or in the vicinity of. When the term "about" is used in connection with a numerical range, it defines that range by extending the boundaries above and below the recited numerical value. In general, the term "about" is used herein to limit a numerical value to a variation of 20%, preferably plus or minus 10% (higher or lower) above and below the specified value. 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 a temperature or molecular weight value), the exact value (i.e., without "about") is preferred.

[0026] The term "comprises and / or comprising" when used in this specification, specifies the presence of the recited features, integers, steps, operations, elements, and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0027] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) means that the scope of the claim is to be interpreted to cover the specified materials or steps recited in the claim and those that do not materially alter one or more of the basic and novel features of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising".

[0028] As used herein, the term transgenic "event" refers to a recombinant plant produced by transforming and regenerating a single plant cell with heterologous DNA (e.g., an expression cassette comprising a gene of interest). The term "event" refers to the original transformant containing the heterologous DNA and / or progeny of the transformant. The term "event" also refers to progeny produced by sexual outcross between the transformant and another maize line. Even after repeated backcrossing to a recurrent parent, the inserted DNA and flanking DNA from the transformed parent are present at the same chromosomal location in the hybrid progeny. Typically, transformation of plant tissue produces multiple events, each of the above events representing insertion of the DNA construct into a different location in the genome of the plant cell. Specific events are selected based on the expression of the transgene or other desired traits.Non-limiting examples of such transgenic events of the present invention include "Event Bt11", which contains the cry1Ab and pat genes and is described in US 6114608 (also referred to as "Bt11 Event" or just "Bt11"); "Event 5307", which contains the eCry3.1Ab and PMI genes and is described in US 8466346 (also referred to as "5307 Event" or just "5307"); "Event MIR604", which contains the mCry3A and PMI genes and is described in US 7361813 (also referred to as "MIR604 Event" or just "MIR604"); "Event MIR162", which contains the Vip3A and PMI genes and is described in US 8232456 (also referred to as "Event MIR162" or just "MIR162"); "Event GA21", which contains the dmEPSPS gene and is described in US 6566587 (also referred to as "GA21 Event" or just "GA21"); "Event 3272", which contains the α-amylase 797E and PMI genes and is described in US 7635799 (also referred to as "3272 Event" or just "3272"); "Event MON810", which contains Cry1Ab and is described in US 6713259 (also referred to as "MON810 Event" or just "MON810"); "Event MON89034", which contains the Cry1A.105 and Cry2Ab genes and is described in US 8062840 (also referred to as "MON89034 Event" or just "MON89034"); "Event TC1507", which contains Cry1F and PAT genes and is described in US 7288643 (also referred to as "TC1507 Event" or just "TC1507"); "Event DAS59122", which contains Cry34 / Cry35 and PAT genes and is described in US 7323556 (also referred to as "DAS59122 Event" or just "DAS59122") and "Event DP4114", which contains Cry1F, Cry34 / Cry35 and PAT genes and is described in US 9790561 (also referred to as "DP4114 Event" or just "DP4114").

[0029] The term "isolated" nucleic acid molecule, polynucleotide or toxin is a nucleic acid molecule, polynucleotide or toxic protein that is no longer in its natural environment. The isolated nucleic acid molecules, polynucleotides or toxins of the present invention can exist in a purified form or can be present in a recombinant host, such as a transgenic bacterial cell or a transgenic plant.

[0030] As used herein, the general term "mass spectrometry" refers to any suitable mass spectrometry method, device 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 device measures the molecular weight of a molecule (as a function of the mass-to-charge ratio of the molecule) by measuring the flight path of the molecule through a set of magnetic and electric fields. The mass-to-charge ratio is a physical quantity widely used in the electrodynamics of charged particles. The mass-to-charge ratio of a specific peptide can be calculated in advance by a person skilled in the art. When subjected to the same electric and magnetic fields, two particles with different mass-to-charge ratios will not move along the same path in a vacuum. The present invention particularly includes the use of high performance liquid chromatography (HPLC), followed by tandem MS analysis of the peptide. In "tandem mass spectrometry", alternative peptides can be filtered in an MS instrument and subsequently fragmented to produce one or more "transition ions", which are analyzed (detected and / or quantified) in a second MS program.

[0031] A detailed overview of mass spectrometry methods and devices can be found in the following references, which are hereby incorporated 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: John Wiley & Sons, pages 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: John Wiley & Sons, pages 10.22.1 - 10.22.24; and Domon and Aebersold (2006) Science 312(5771):212 - 17.

[0032] A peptide is a short polymer formed by the sequential linking of α-amino acids. Peptides can also be produced by digesting polypeptides (such as proteins) with proteases.

[0033] "Plant" means any plant at any stage of development, particularly seed plants.

[0034] "Plant cell" is the structural and physiological unit of a plant, containing a protoplast and a cell wall. Plant cells can be in the form of isolated single cells or cultured cells, or as part of a higher organizational unit (such as, for example, plant tissues, plant organs, or whole plants).

[0035] "Plant cell culture" means a culture of plant units (such as, for example, protoplasts, cultured cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at different developmental stages).

[0036] "Plant material" refers to leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0037] "Plant organ" is a distinct and visible structured and differentiated part of a plant, such as a root, stem, leaf, bud, or embryo.

[0038] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. It includes any plant tissue in a plant or in culture. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The combined or separate application of this term with any specific type of plant tissue listed above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[0039] As used herein, the term "surrogate peptide" refers to a peptide derived from a target protein by proteolytic digestion (e.g., trypsin digestion), which functions in a mass spectrometry assay to generate one or more transition ions that, when the target protein is co-present with one or more other proteins and / or transgenic proteins in a complex biological matrix (such as a sample from a transgenic plant), differentially detect and / or quantify the target protein in combination with the surrogate peptide, and do not detect and / or quantify the one or more other proteins or transgenic proteins in the biological matrix. A "surrogate peptide" may also be referred to as a "signature peptide" of the target protein. For example, the HPPD surrogate peptides of the present invention generate one or more transition ions that, when the HPPD protein is co-present with one or more non-HPPD proteins, differentially detect and / or quantify the target HPPD protein in a complex biological matrix in combination with the HPPD-surrogate peptide. According to embodiments 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.

[0040] A "labeled surrogate peptide" is a non-naturally occurring surrogate peptide that is labeled such that it is readily detectable in a mass spectrometry assay. For example, the label can be a stable isotope-labeled amino acid (SIL), such as lysine, isoleucine, valine, or arginine. Thus, an SIL-labeled surrogate peptide has the same amino acid sequence as the unlabeled 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 can be designated as GNFSELFK[C13N15-K], etc.

[0041] As used herein, the term "stacked" refers to the presence of multiple heterologous polynucleotides or transgenic proteins or transgenic events incorporated in the genome of a plant.

[0042] As used herein, "target protein" means a protein that is intended to be selectively detected and / or quantified by a labeled surrogate peptide when the target protein is in a complex biological matrix.

[0043] Nucleotides are represented herein by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise represented 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; I), 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).

[0044] The present invention encompasses compositions, methods, and systems useful for performing mass spectrometry for differential detection and / or quantification of one or more target HPPD proteins in complex biological samples derived from crop plants, which complex biological samples (e.g., biological samples from leaves, stems, roots, pollen, and seeds of one or more crop plants) contain a mixture of target and non-target proteins, and each biological sample may have a different impact on the mass spectrometry results.

[0045] The accuracy of quantifying target proteins by multiple reaction monitoring assays (MRM) of mass spectrometry depends entirely on the selection of appropriate surrogate peptides and the ability of the surrogate peptide / transition ion combinations to distinguish the target proteins. Many different combinations of the surrogate peptides of the present invention can be simultaneously monitored and quantified by MRM assays using one or more specific surrogate peptides from the HPPD proteins, and thus provide a method for identifying and quantifying each target HPPD protein within a given biological sample by mass spectrometry. The available surrogate peptides that make up the kit can be analyzed individually or in any combination in a single MRM assay, or in multiple MRM assays.

[0046] The surrogate peptides of the present invention in combination with MRM-based assays have numerous applications, including quantitative peptide / protein analysis for determining expression levels at different growth stages, determining potential exposure levels for environmental risk assessment, determining different levels of target proteins in food processing, determining expression levels in comparative studies, and comparing expression levels in generational studies. In the broadest sense, these unique surrogate peptides of the target proteins can be used in combination with MRM assays for monitoring or quantifying herbicide tolerance traits that may occur in crop plants or transgenic events, or breeding stacks of multiple transgenic events within specific tissues (i.e., leaves, roots, nuclei, pollen).

[0047] 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 the characteristic peak areas to each other. The relative levels of individual HPPD surrogate peptides can be quantified from different sample or tissue types. Typically, the relative quantitative levels are determined by comparing the peptide abundances in the MRM measurements to stable isotope-labeled (SIL) synthetic peptide analogs that serve as internal standards for each target surrogate peptide. Contrary to what is commonly taught in the art, the SIL peptides are labeled by incorporation of 13 C6 15 N2] lysine or 13 C6 15 N4] arginine, but may also include other amino acids such as isoleucine and valine. The SIL standards need to be of high purity and should be quantitatively standardized by amino acid analysis. Contrary to what is commonly taught in the art, the SIL of the present invention is spiked into the sample immediately after protein digestion and thus used to correct subsequent analysis steps. The SIL co-elutes with the unlabeled surrogate peptide in the liquid chromatography separation and shows the same MS / MS fragmentation pattern, but only differs in mass due to the isotope labeling. The mass shift of the labeled surrogate peptide and product ions results in the mass spectrometer differentiating between the unlabeled and labeled peptides. Since complex peptide digests typically contain multiple sets of co-eluting transitions that may be mistaken for the target peptide, the co-elution of the isotope-labeled standard can identify the correct signal and provide optimal protection against false positive quantification. Since a known concentration of spiked SIL standard is spiked into each sample, the relative amounts of each corresponding surrogate peptide from different target proteins of the HPPD protein can be determined. Since the relative quantification of a single peptide or multiple peptides can be made relative to the amount of another peptide or multiple peptides within or between samples, the relative amounts of multiple peptides present can be determined by determining whether the peak areas within a biological sample are related to each other. The relative quantitative data between different samples derived from the individual characteristic peak areas are typically normalized to the amount of protein analyzed per sample. Relative quantification of multiple peptides from multiple proteins can be performed simultaneously in one sample and / or multiple samples to further understand the relative protein amounts of one peptide / protein relative to other peptides / proteins.

[0048] The absolute quantification level of HPPD can be determined by MRM-based assays by comparing the characteristic peak areas of individual surrogate peptides of the corresponding protein from a biological sample with one or more internal standards of known amounts 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 protein. Multiple reaction monitoring (MRM) assays involve weighing a non-target sample of the target protein with an accurately spiked concentration; extracting the sample in lysis buffer and homogenizing it; centrifuging the sample to separate soluble and insoluble proteins to enrich and reduce the complexity of the extract; digesting the soluble protein sample with trypsin (tissues or biological samples can be treated with one or more proteases, including but not limited to trypsin, chymotrypsin, pepsin, endoproteinase Asp-N, and Lys-C, for a period of time to fully digest the sample), centrifuging the sample, adding a fixed concentration of SIL peptides (in absolute quantification, SIL is used as an indicator); desalting by solid-phase extraction using cation exchange to minimize matrix effects or interferences and reduce ion suppression; and analyzing the sample by liquid chromatography tandem mass spectrometry. Typically, an ion trap mass spectrometer or another form of mass spectrometer capable of global analysis (for identifying as many peptides as possible from a single complex protein / peptide lysate) is commonly used for analysis. Although MRM-based assays can be developed and performed on any type of mass spectrometer, the triple quadrupole instrument platform is generally considered the most favorable instrument platform for MRM assays. Measure the target surrogate peptides and SIL unique to seven proteins by LC-MS / MS. Determine the peak area ratio of each target peptide (peak area of the surrogate peptide / peak area of the corresponding SIL peptide). Use the peak area ratio to back-calculate the concentrations of the seven target proteins from the calibration curve. Absolute quantification can be performed on many peptides, which allows for the simultaneous quantitative determination of multiple proteins in a single sample and / or multiple samples to understand the absolute protein content in a single biological sample or a large group of samples.

[0049] In some embodiments, the present invention encompasses a labeled alternative peptide that functions in mass spectrometry to selectively detect or quantify 4-hydroxyphenylpyruvate dioxygenase (HPPD) protein in a protein mixture in one or more biological samples from one or more crop plants, the alternative peptide comprising 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 aspects, the labeled alternative peptide is labeled by incorporation of stable isotope labeled (SIL) amino acids. In other aspects, the SIL amino acids are lysine, isoleucine, valine or arginine. In other aspects, the crop plant is barley, rice, soybean, wheat, oats or maize. In additional aspects, the crop plant is barley, rice, soybean, wheat or rice, and the alternative peptide comprises a label and the amino acid sequence of SEQ ID NO:1. In additional aspects, the crop plant is maize, and the alternative peptide comprises a label and the amino acid sequence of SEQ ID NO:2.

[0050] In some embodiments, the present invention encompasses an assay kit comprising at least two labeled alternative peptides, the alternative peptides comprising an amino acid sequence selected from the group consisting of GNFSELFK (SEQ ID NO:1) and GNFSQLFK (SEQ ID NO:2).

[0051] In some embodiments, the present invention encompasses a method for simultaneously detecting or quantifying one or more target HPPD proteins in a complex biological sample containing a mixture of the target protein and non-target proteins from a crop plant, the method comprising: (a) obtaining a biological sample from the crop plant; (b) extracting proteins from the biological sample to obtain an extract containing a protein mixture; (c) reducing the amount of insoluble proteins in the extract of step b to obtain a concentrated extract of soluble proteins; (d) digesting the soluble proteins in the extract of step c to obtain an extract containing peptide fragments, wherein the peptide fragments contain at least one alternative peptide specific for the target protein; (e) concentrating the peptide fragments in the extract of step d; (f) adding one or more labeled alternative peptides of the present invention, wherein each labeled alternative peptide has the same amino acid sequence as each alternative peptide of the target protein, and wherein the number of labeled alternative peptides added is equal to the number of target proteins in the mixture; (g) concentrating the alternative peptides and the labeled alternative peptides by reducing the amount of non-alternative peptides in the mixture; (h) resolving the peptide fragment mixture from step g by liquid chromatography; (i) analyzing the peptide fragment mixture obtained from step h by mass spectrometry, wherein the detection of the transition ion fragments of the labeled alternative peptide indicates the presence of the target protein from which the alternative peptide is derived; and, optionally, (j) calculating the amount of the target protein in the biological sample by comparing the mass spectrometry signal generated by the transition ion fragments of step i with the mass spectrometry signal generated by the transition. In some aspects, the crop plant is barley, rice, soybean, wheat or rice, and the alternative peptide contains a label and the amino acid sequence of SEQ ID NO:1. In other aspects, the crop plant is maize, and the alternative peptide contains a label and the amino acid sequence of SEQ ID NO:2. In other aspects, the peptide is labeled by incorporation of a stable isotope-labeled (SIL) amino acid. In additional aspects, the SIL amino acid is lysine, isoleucine, valine or arginine.

[0052] Many references in the art have proposed many different methods to predict which alternative peptides are most suitable for any given target protein, and many references have proposed shortcuts for quantifying target proteins using mass spectrometry, such as Mead et al. 2009. Mol. Cell. Proteomics [Molecular and Cellular Proteomics] 8:696-705 and U.S. Patent No. 8,227,252. However, relying on such prediction methods and shortcuts can lead to confounding results because unpredictable factors can interfere with mass spectrometry-based assays, resulting in decreased sensitivity and inaccurate quantification. At least one major factor lies in the biological matrix itself. For example, it is highly unpredictable and difficult to identify a single transition ion from alternative peptides that are equally effective in biological samples from leaves, roots, pollen, and seeds of crop plants. Differences in the chemical composition, pH, or ionic strength of the matrix can affect proteolysis, peptide stability, aggregation, or ionization in the MS instrument. Therefore, alternative peptides and specific alternative peptide / transition ion combinations for all relevant matrices (especially those of crop plants) must be identified and empirically tested to overcome the unpredictable nature of such assays. The present invention employs a two-step method in developing a mass spectrometry assay for specifically detecting and / or quantifying a target protein, comprising: 1) testing and selecting alternative peptides from a peptide library derived from proteolytic cleavage of the target protein, and testing combinations of SIL alternative peptides and transition ion peptides, and selecting combinations that specifically detect and quantify the target protein in all biological matrices (such as biological samples from leaves, roots, pollen, or seeds of crop plants); and 2) empirically determining appropriate methods for sample preparation and mass spectrometer conditions for all alternative peptides and alternative peptide / transition ion combinations in all biological matrices (including leaves, roots, pollen, and seeds of crop plants, especially maize plants).

[0053] Accordingly, in some embodiments, the present invention encompasses a method for simultaneously detecting and / or quantifying one or more target proteins in a complex biological sample comprising a mixture of a target transgenic protein and a non-transgenic protein from a transgenic plant, the method comprising the steps of: a) obtaining a biological sample from the transgenic plant; b) extracting proteins from the biological sample to obtain an extract comprising a protein mixture; c) reducing the amount of non-transgenic insoluble proteins in the extract of step b to obtain a concentrated extract of soluble proteins; d) digesting the soluble proteins in the extract of step c to obtain an extract comprising peptide fragments, wherein the peptide fragments comprise at least one unlabeled surrogate peptide specific for 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 unlabeled surrogate peptide derived from the target protein, and wherein the number of labeled surrogate peptides added is equal to the number of target proteins in the mixture; g) concentrating the unlabeled surrogate peptides and the labeled surrogate peptides by reducing the amount of non-surrogate peptides in the mixture; h) resolving the mixture of peptide fragments from step g by liquid chromatography; i) analyzing the mixture of peptide fragments obtained from step h by mass spectrometry, wherein detection of a transition ion fragment of an unlabeled surrogate peptide indicates the presence of the target protein from which the surrogate peptide is derived; and optionally j) calculating the amount of the target protein in the biological sample by comparing the mass spectrometry signal generated by the transition ion fragment of step i with the mass spectrometry signal generated by the transition ion of the labeled surrogate peptide.

[0054] The following specific examples are included to illustrate the preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present invention, and thus can be considered to constitute preferred modes of its practice. However, in light of this disclosure, those skilled in the art will appreciate that many changes can be made in the specific embodiments disclosed and still obtain a similar or like result without departing from the concept, spirit, and scope of the present invention. More specifically, it is apparent that certain chemically and physiologically related agents can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications that are obvious to those skilled in the art are deemed to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0055] Examples

[0056] Although the present invention has been described in connection with specific embodiments thereof, it is to be understood that the apparatus of the present invention is capable of further modification. This patent application is intended to cover any variations, uses, or adaptations of the present invention generally following the principles of the present invention, including such departures from the present disclosure as come within known or customary practice in the art to which the present invention pertains and as may be applied to the key features hereinbefore set forth and fall within the scope of the appended claims.

[0057] All publications and patent applications mentioned in this specification indicate the state of the art of those skilled in the art to which the present invention pertains. All publications and patent applications are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0058] Example 1. Validation of the Quantification of Endogenous 4-Hydroxyphenylpyruvate Dioxygenase in Commodity Crop Articles of Commerce

[0059] The objective of this study was to validate a mass spectrometry (MS)-based method for the quantification of 4-hydroxyphenylpyruvate dioxygenase (HPPD) protein in various commodity crop articles of commerce (barley, maize, rice, soybean, and wheat seeds, and barley, maize, oat, soybean, and wheat feeds) using liquid chromatography tandem mass spectrometry (LC-MS / MS). Specific surrogate peptides unique to the HPPD protein were used to determine relative concentrations. Prior to implementing the quantification method in the study, the method was validated for the intended use according to Good Laboratory Practice Standards (GLPS). Commodity crop samples were used to evaluate method performance parameters, including specificity, linearity, limit of quantification, carryover, precision and accuracy, extraction efficiency, and stability.

[0060] The assessment of specificity showed no significant interference in the retention times of the unlabeled peptides (average ratio difference between two transitions of blank and buffer < 30.0%) and the stable isotope-labeled (SIL) peptides (SIL signal of QC0 (endogenous) ≤ 5.0%) in any of the tested commodity crop samples. All specificity parameters met the acceptance criteria.

[0061] The lower limit of quantification (LLOQ) of each characteristic peptide used as a surrogate for the target HPPD protein was determined. This limit of quantification was: 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.

[0062] The validated quantification ranges (LLOQ) and upper limits of quantification (ULOQ) of HPPD for each commodity crop are summarized in Table 1.

[0063] Table 1. Quantification ranges

[0064]

[0065] The within - batch and between - batch CV of QC0 samples was ≤25.0%, and the CV of quality control (QC) samples at three concentrations (low, medium, and high) was ≤20.0%, indicating good method precision. Similarly, the within - batch and between - batch deviation % of low, medium, and high QC samples was within ±20.0%, indicating good method accuracy. Therefore, all method precision and accuracy parameters met the acceptance criteria. The within - batch and between - batch precision and accuracy of the method are summarized in Table 2.

[0066] Table 2. Within - batch and between - batch precision and accuracy ranges of HPPD in commodity crops

[0067]

[0068] A linear equation with a data weight of 1 / x was used to represent the concentration / detector response relationship. Therefore, all established data met the acceptance criteria and were considered suitable for linear evaluation.

[0069] 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 extraction for all commodity crops was below 20.0% except for wheat seeds, where the CV was 27.5%. However, the precision and accuracy results (QC0) from single - extraction wheat seeds indicated that a precision of ≤20.0% was achieved.

[0070] The processing stability (dry extract) was evaluated at -20 °C for 6 days. The stability evaluation met the acceptance criteria. For all commercial crops, CV ≤ 25.0%, and the difference % in the peak area ratio between stability and day 0 QC was within ±25.0%.

[0071] All evaluated performance parameters met the specified acceptance criteria, except for the final extraction % of barley and wheat seeds being 8.2% and 6.2% respectively, and the precision of the first round of extraction of wheat seeds being 27.5% CV. Since the concentration of HPPD in the sample analysis will be determined by a single extraction (first round) and adjusted according to the extraction efficiency %, and the precision of wheat seeds in the precision and accuracy runs reached ≤ 20.0%, it has no impact on the results of the study. Based on the results of this study, the mass spectrometry-based method has been proven applicable for the quantification of HPPD protein in commercial crops according to GLPS.

[0072] The aim of this study was to validate a mass spectrometry (MS)-based method for the quantification of 4-hydroxyphenylpyruvate dioxygenase (HPPD) protein in various commercial crops (barley, maize, rice, soybean, and wheat seeds, as well as barley, maize, oat, soybean, and wheat feeds) using liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis. Specific surrogate peptides for HPPD protein were used to determine the relative concentrations in different plant species / matrices (Table 3). Before implementing the quantification method in the study, the method was validated for the intended use according to Good Laboratory Practice Standards (GLPS). Commercial crop samples were used to evaluate method performance parameters, including specificity, linearity, limit of quantification, residue, precision and accuracy, extraction efficiency, and stability.

[0073] Table 3. HPPD surrogate peptides

[0074]

[0075] The commercial crop materials used in this study were matrices from various plant species, which were used to prepare linear samples and quality control (QC) samples. Table 4 identifies the sources of the matrix materials. These samples were provided by the sponsor and stored at a nominal temperature of -80 °C ± 10 °C until use.

[0076] Table 4. Commercial crop materials

[0077]

[0078] Standards and QC samples

[0079] For linearity evaluation, a reverse curve was generated. A set of eight non-zero standards (STD), including the LLOQ (STD 1) and ULOQ (STD 8) STD, were prepared using commercial crop extracts fortified with SIL peptides at different concentrations.

[0080] QC samples were prepared using a commercial crop extract fortified with an unlabeled peptide at three different concentrations (low, medium, and high). Tables 5 and 6 list the nominal concentrations.

[0081] Table 5. Concentrations of standards in commercial crops (nominal)

[0082]

[0083] a DW - dry weight

[0084] Table 6. Concentrations of QC samples in commercial crops (nominal a )

[0085]

[0086] a Nominal = nominal concentration before adjustment with QC0 (endogenous level of unlabeled peptide)

[0087] Synthetic peptides

[0088] The purified and quantified SIL and unlabeled synthetic peptides used in this study are listed in Table 7. The synthetic peptides were provided by JPTPeptide Technologies GmbH and stored at a nominal temperature of -20 °C until use.

[0089] Table 7. List of synthetic peptides

[0090]

[0091] Analytical method

[0092] The validation process of an LC-MS / MS-based method to determine the quantity of HPPD protein in commercial crops involves the following experimental and data evaluation steps. Briefly, the LC-MS / MS method used to identify and quantify HPPD surrogate peptides in a complex biological matrix includes: (i) weighing the lyophilized commercial crop; (ii) homogenizing / extracting proteins from the plant species / matrix sample in lysis buffer; (iii) centrifuging the sample to separate soluble and insoluble proteins to enrich the target protein and reduce sample complexity; (iv) digesting the soluble protein sample with trypsin; (v) centrifuging the sample; (vi) adding SIL peptides (fixed or variable, depending on the evaluation); (vii) desalting by solid-phase extraction using cation exchange to minimize matrix effects or interferences and reduce ion suppression; and (viii) analyzing the sample by LC-MS / MS. The target surrogate peptides unique to each commercial crop HPPD protein and the corresponding SIL peptides are measured by LC-MS / MS. Using MultiQuant TMAnalyze the data of software version 3.0.2, where the chromatographic peak areas of each alternative peptide (unlabeled) and each corresponding SIL peptide for each sample are integrated.

[0093] For the reverse curve, determine the peak area of the SIL peptide for each alternative peptide. Then, for each alternative peptide, plot the peak area of the SIL peptide of each standard sample on the y-axis as a function of the protein concentration (x-axis) to create the reverse curve.

[0094] For the QC samples, the peak area ratio (peak area of the 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:

[0095]

[0096] Sample processing

[0097] Weigh each commercial crop between 10 and 18 mg and add it to a test tube containing matrix A lysis beads (MP Biomedicals). Then add the lysis buffer (0.1% RapiGest in phosphate buffered saline (PBS)) TM (Waters) to each sample tube and homogenize using a homogenizer (MP Biomedicals). Then centrifuge the samples at 4 °C to remove insoluble materials. Transfer the supernatant as described in Table 8; combine and dilute in 0.1% RapiGest in PBS. These diluted commercial crops were fortified with either SIL peptides at different concentrations to obtain standards or with unlabeled peptides to obtain QC samples.

[0098] Table 8. Matrix dilution factors

[0099]

[0100] For each processed standard and QC sample, add an equal volume of 2,2,2-trifluoroethanol (TFE) to denature the protein, then dilute the TFE to 10% with 100 mM ammonium bicarbonate and digest with 0.1 μg / μL trypsin and incubate at 37 °C for 14 to 18 hours. After digestion, acidify the samples with formic acid (FA) at a final concentration of 5%, and add SIL peptides at variable or fixed concentrations to each sample according to the validation assessment except for the blanks and residual blanks. Then desalt the samples by solid-phase extraction using a mixed-mode cation exchange (MCX) μElution plate. Collect the eluate and transfer it to two MS plates, evaporate it to dryness and store it at -20 °C (nominal) until MS analysis.

[0101] The processed samples were redissolved in 11 μL of 92.5 / 7.5 water / acetonitrile (ACN) + 0.2% FA, followed by sonication, vortexing, and centrifugation. For each sample, eight microliters of the material was injected into a NanoAcquity ultra performance liquid chromatography (Waters Corporation) interfaced with a QTRAP 6500 mass spectrometer (AB Sciex). 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 gradient used is shown in Table 9 below. The flow rate was 28.000 μL / min. Analytes were measured in positive ion mode using a Turbo V MS source. Data acquisition was performed using version 1.6 (AB Sciex).

[0102] Table 9. LC gradient for LC-MS / MS determination

[0103]

[0104] a DMSO - dimethyl sulfoxide

[0105] The surrogate peptides and the corresponding SIL peptides are unique to the HPPD protein. Chromatographic peak areas of each surrogate peptide (unlabeled) and each corresponding SIL peptide for each sample were integrated using MultiQuant version 3.0.2 (AB Sciex).

[0106] For the reverse curve, the peak area of the SIL peptide was determined 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 create the reverse curve.

[0107] For the QC samples, 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 samples was calculated as described above.

[0108] Using Microsoft Office software, mean, standard deviation (SD), and coefficient of variation (CV) calculations were performed. Unrounded values were used for the mean, SD, and CV calculations and then appropriately rounded in the summary table.

[0109] Specificity

[0110] Specificity was determined by the ability of the method to measure and distinguish surrogate peptides in the presence of various plant matrix components. Specificity was measured by determining the presence or absence of each unique surrogate peptide of the HPPD protein by LC-MS / MS.

[0111] For all commodity crop extracts described below, the assay specificity was evaluated using quantifier and qualifier transitions for unlabeled peptides and quantifier transitions only for SIL peptides.

[0112] Unlabeled peptides

[0113] The following analysis was performed:

[0114] The assay specificity was evaluated by comparing the average ratio of two transitions (quantitative / qualitative) monitored in digested bovine serum albumin (BSA) buffer fortified with unlabeled peptides (n = 3) with blank (n = 3). The blank was considered as the matrix containing endogenous HPPD protein treated with undetectable amounts of unadded SIL or unlabeled peptides.

[0115] The difference % was calculated using the following formula:

[0116]

[0117] The following acceptance criteria were used to confirm the applicability of the specificity assessment for unlabeled peptides: The difference % between the blank and the buffer must be within 30.0%.

[0118] SIL peptides

[0119] The following analysis was performed:

[0120] The assay specificity was evaluated by determining the peak areas of SIL peptides (quantifier transition) in QC0 (endogenous, n = 3) and blank (n = 3). The blank was considered as the matrix containing endogenous HPPD protein treated with undetectable amounts of unadded SIL or unlabeled peptides.

[0121] The % of QC0 (endogenous) was calculated using the following formula:

[0122]

[0123] The following acceptance criteria were used to confirm the applicability of the specificity assessment for SIL peptides: The average peak area of SIL peptides in the blank sample must be ≤ 5.0% of the average peak area of SIL peptides in the QC0 (endogenous) sample.

[0124] Linearity

[0125] Linearity is the ability of the method to yield results that are mathematically defined by the amount of analyte in the sample and the responsiveness. Linearity is evaluated based on the accuracy of the method. For HPPD in each plant species / matrix, the simplest regression model defining the inverse curve, i.e., linear curve fitting, is used. The model is applied based on the goodness of fit using the correlation coefficient (R value).

[0126] A data weight of 1 / x is applied to all peptides. The same regression model and the same data weight are applied to all assay runs for each plant species / matrix.

[0127] The following run acceptance criteria are used to confirm the applicability of the linearity evaluation: (1) At least 75.0% of the non-zero standards must be valid, and the deviation must not exceed 20.0% of the nominal concentration, except that the deviation of the LLOQ must not exceed 25.0%; (2) The interpolated curve must have R ≥ 0.9900.

[0128] Limit of Quantitation

[0129] LLOQ and ULOQ are the minimum and maximum concentrations at which the response of the surrogate peptide can be determined within the acceptable accuracy limits. The LLOQ and ULOQ of the surrogate peptide in each plant species / matrix are determined using the said inverse curve.

[0130] The following acceptance criteria are used to confirm the applicability of the LLOQ and ULOQ evaluations: The LLOQ and ULOQ standards must meet the accuracy criteria defined above.

[0131] The quantitative range of HPPD is the interval between the upper and lower concentration limits for which the analytical procedure has been demonstrated to have suitable levels of accuracy and linearity (see the standard concentrations in Table 5). Using the data obtained from the linearity evaluation of the method, the quantitative range of HPPD in each plant species / matrix is determined. Determining the accuracy effectively validates the high and low concentrations tested as the quantitative range of the procedure.

[0132] Residue refers to the presence of the analyte in subsequent injections. In this study, residue during linearity evaluation was determined by assessing two blank samples injected after each ULOQ standard. They must be free from interference. The following run acceptance criteria were used to evaluate residue for all linear runs. Evaluate the first residual blank sample injected after the ULOQ standard. The second residual blank was evaluated but was not part of the acceptance criteria. At least 50% of the first residual blank sample injected after the ULOQ standard must be within the acceptable interference range as described below, with retention times: the peak area of the SIL peptide must be ≤ 20.0% of the average peak area of the SIL peptide in the LLOQ standard. Additionally, in the precision, accuracy, and stability evaluations, reconstitution buffer (RSB) samples were analyzed to assess residue after injection of high QC samples. The order of evaluating residue was: high QC, then two RSB samples.

[0133] Run acceptance criteria

[0134] As part of validation, precision, accuracy, and stability runs included QC samples to demonstrate run acceptance. The following criteria must be met for the above runs to be considered valid. Each plant species / matrix was treated independently to meet the acceptance criteria.

[0135] QC samples

[0136] QC samples provided the basis for accepting or rejecting a run. QC samples were prepared by fortifying the sample matrix with unlabeled peptides at known concentrations. The following acceptance criteria were used to confirm the suitability of QC samples using low QC (QC1), mid-range QC (QC2), and high QC (QC3): (1) at least 50% of the low, medium, and high concentration QC samples must be within ±20.0% deviation; (2) at least 67% of the low, medium, and high concentration QC samples must be within ±20.0% deviation of their nominal values.

[0137] Precision and accuracy

[0138] Precision is the degree of agreement between individual test results when the procedure is repeatedly applied to multiple samplings of a homogeneous sample. Accuracy is the degree of agreement between the values found and the acceptable reference value when the procedure is repeatedly applied to multiple samplings of a homogeneous sample. In this study, the precision and accuracy of the HPPD protein in each plant species / matrix determined by LC-MS / MS were determined by evaluating the variability between assays and between analysts.

[0139] The precision and accuracy of the method were evaluated using QC samples. Precision and accuracy determinations were performed using QC samples at three concentrations (low, medium, and high) (Tables 5 and 6). The QC samples were prepared from commercial crop extracts and fortified with unlabeled peptides.

[0140] Precision (CV) and accuracy (bias) were evaluated by two different analysts on three different days in three independent assay runs.

[0141] Each precision and accuracy run consisted of three replicates of each QC concentration. QC0 (endogenous) was also included and analyzed in triplicate.

[0142] The percent bias was calculated using the following formula:

[0143]

[0144] Runs of precision and accuracy that met the run acceptance criteria specified in Section 0 were used to evaluate within-batch precision and accuracy. Then, runs of precision and accuracy that met the within-batch criteria were used to evaluate between-batch precision and accuracy. The following acceptance criteria were used to confirm the applicability of the precision and accuracy evaluations for within-batch and between-batch runs: (1) The CV of the QC0 sample must be ≤ 25.0%; (2) The CV of the low, medium, and high concentration QC samples must be ≤ 20.0% and the bias must be within ±20.0%.

[0145] In addition, the maximum injection time length for the assay runs used to analyze study samples was established by looking at the batch sizes of the precision and accuracy runs. The runs of precision and accuracy with the maximum batch size were used to set the maximum injection time length for the analysis of study samples. In addition, a trend analysis was performed on the QC0 (endogenous) samples.

[0146] The extraction efficiency is the amount of the target protein (i.e., HPPD) recovered from the matrix. The extraction efficiency was determined by sequential extraction. If the total protein recovered in the last round did not exceed 5.0%, the extraction recovery was considered final. The efficiency of the protein extraction method was evaluated by repeated extraction of HPPD using commodity crops.

[0147] One analyst extracted three replicates of each commodity crop. The insoluble material was collected and re-extracted 3 times, and each supernatant was retained for analysis.

[0148] The extraction efficiency for each sample was calculated using the following formula:

[0149]

[0150] The following acceptance criteria were used to confirm the applicability of the extraction efficiency for each protein: Sequential extraction was considered complete when the final extraction result did not exceed 5.0% of the total recovered material for each individual protein from all combined extractions:

[0151]

[0152] The extraction efficiency (repeated mean of the first iteration) is expected to be ≥ 60.0% recovery, and the precision of the first round of extraction must be ≤ 20.0%.

[0153] Stability

[0154] The stability of the HPPD protein in a given plant species / matrix over a given time interval under given conditions was evaluated. The stability of the HPPD protein in each plant species / matrix, as well as the stability of the surrogate peptides in the processed samples, was determined.

[0155] Stability of the processed samples

[0156] The stability of the processed samples of the dry extracts was analyzed at two different concentrations (low and high), with each concentration repeated three times. Freshly prepared and processed QC samples were analyzed by LC-MS / MS (recognized as Day 0). The dried stability samples were not immediately analyzed by LC-MS / MS but were stored for a predetermined period of time.

[0157] The dried stability samples were stored at a nominal temperature of -20 °C for 6 days (146 hours 16 minutes), then re-dissolved and analyzed by LC-MS / MS.

[0158] The following acceptance criteria were used to confirm the stability of the surrogate peptides in the processed samples: (1) The overall average peak area ratio of the Day 0 samples and the stability samples after processing must be ≤ 25.0% CV; (2) The percentage difference in the peak area ratio between the stability QC and the Day 0 QC (processed on the day of injection) must be within ±25.0%.

[0159]

[0160] According to the validation protocol, the residual assessment was appropriately outlined in the general description, which stated that the peak area was used for the residual assessment. However, by oversight, the description in the acceptance criteria stated the peak area ratio. The peak area is the correct method for the assessment, so the peak area was used to evaluate the interference in this study. Therefore, since the appropriate method for evaluating the interference in the residual blank (i.e., the peak area) was applied, there was no impact on the data quality and integrity.

[0161] According to the validation protocol, the peak area ratio (SIL / unlabeled) of each peptide for quantitative conversion was used to plot the calibration curve data on a linear scale. However, instead, the peak area of each peptide for quantitative conversion was used for plotting. Due to the contribution of the unlabeled signal from the stable isotope-labeled peptide at high spiked concentrations, the peak area ratio cannot be used for linear evaluation. Since the linear evaluation was performed, there was no impact on the data quality and integrity.

[0162] According to the validation protocol, the extraction efficiency assessment was carried out using 4 consecutive extractions (cycles). For the two matrices (i.e., barley and wheat seeds), the extraction efficiency results did not meet the following acceptance criteria.

[0163] Barley seeds - The result of the final extraction (4th cycle) was 8.2% (acceptance criterion: < 5.0%)

[0164] Wheat seeds - The result of the final extraction (4th cycle) was 6.2% (acceptance criterion: < 5.0%).

[0165] The precision of the first extraction was 27.5% CV (acceptance criterion: ≤ 20.0%).

[0166] However, it had no impact on the study results because: (1) The final extraction results of barley and wheat seeds showed that after four extractions, the extraction of HPPD protein was nearly complete. In addition, the concentration of HPPD in the sample analysis would be determined by a single extraction (the first cycle) and adjusted according to the extraction efficiency %. And (2) For wheat seeds, the precision of the first extraction (27.5% CV) exceeded the 20.0% CV standard, but the precision and accuracy results of QC0 (endogenous) from three validation runs (runs 4 to 6) (from single extractions) showed that a precision of ≤ 20.0% was achieved.

[0167] According to the validation protocol, for each precision and accuracy run, two (2) RSB samples were injected after the high QC sample at the end of each matrix. However, due to negligence, for one matrix (i.e., wheat seeds), the RSB sample injected after the high QC sample (QC3 replicate 3) was obtained using the MRM method for the peptide GNFSQLFK (SEQ ID NO:2) instead of the peptide GNFSELFK (SEQ ID NO:1). Therefore, the RSB residue assessment for wheat seeds was not carried out.

[0168] However, it had no impact on the study results because: (1) No residue of the peptide GNFSELFK (SEQ ID NO:1) was observed in any other matrix (including wheat feed), so the overall conclusion was that the peptide GNFSELFK (SEQ ID NO:1) did not show any tendency to residue, and it could be inferred that the residue of the peptide GNFSELFK in the wheat seed matrix was highly unlikely; (2) Therefore, the available dataset allowed the establishment of an RSB residue acceptance criterion (i.e., at RT, ≤ 5.0% of the peak area of the unlabeled peptide compared to the peak area of QC0) for sample analysis of all matrices (including wheat seeds) related to the peptide GNFSELFK (SEQ ID NO:1) and GNFSQLFK (SEQ ID NO:2).

[0169] There were also minor SOP deviations, which had no impact on the study. All of these were documented in the study file. No other circumstances occurred during the conduct of this study that would have an adverse effect on the quality or integrity of the generated data.

[0170] Specificity

[0171] The assay specificity of unlabeled peptides and SIL peptides was evaluated in all commodity crop extracts. This evaluation showed some minor interferences in the retention times of the surrogate peptides and the corresponding SIL peptides in the tested commodity crop samples. For unlabeled peptides, in all tested plant species / matrices, the percentage difference in the average ratio of two transitions between blank and buffer samples was less than 30.0%, and all interferences were ≤ 5.0% of the average peak area of the SIL peptides in the QC0 (endogenous) samples, meeting the specificity acceptance criteria.

[0172] Linearity

[0173] The linearity of HPPD protein in each plant species / matrix was determined using the reverse curves described in Sections 3.4 and 0. The simplest regression model defining the reverse curve, i.e., linear regression, was used. A data weight of 1 / x was used for all plant species / matrices. Table 10 summarizes the standard curve parameters (slope, intercept, and R value) for the linearity evaluation. The correlation coefficient (R) of the standard curve of HPPD protein in each plant species / matrix was ≥ 0.9900. All established data met the acceptance criteria described in Part 0 and were considered suitable for linearity evaluation.

[0174] Table 10. Reverse curve parameters of the linear equation with a weighting factor of 1 / x in commodity crops

[0175]

[0176] The standard concentrations of the reverse-calculated reverse curves in each plant species / matrix are provided in Table 11.

[0177] Table 11. Standard concentrations of the reverse-calculated reverse curves in commodity crops

[0178]

[0179] For each standard of each plant species / matrix, N = 2 limits of quantification

[0180] In the linearity evaluation, the accuracy (bias) of the LLOQ and ULOQ samples was within ±25.0% and ±20.0% respectively (Table 11), so the limits of quantification were set as the concentrations of the lowest and highest non-zero standards. Table 12 summarizes the lower and upper limits of quantification of HPPD in each plant species / matrix.

[0181] Table 12. Limits of quantification of HPPD in commodity crops

[0182]

[0183] The quantification range of HPPD in each plant species / matrix is the interval between the lower limit (LLOQ) and upper limit (ULOQ) concentrations, and the analytical procedure has been demonstrated to have an appropriate level of accuracy. The LLOQ and ULOQ samples meet the accuracy criteria for HPPD in each plant species / matrix. The quantification range of the method is set for each plant species / matrix (Table 13).

[0184] Table 13. Quantification range of HPPD in commercial crops

[0185]

[0186] Residue

[0187] After testing the ULOQ standard of the commercial crop samples, the residue is determined by evaluating the injected blank samples in the linearity assessment. The evaluation shows no interference with the retention time of the SIL peptide. In at least 50% of the first residue blank samples injected after the ULOQ standard, the peak area ratio of the SIL peptide is ≤ 20.0% of the average peak area ratio of the SIL peptide of the LLOQ standard, meeting the residue acceptance criteria.

[0188] In addition, the residue is evaluated during precision, accuracy, and stability runs using RSB samples after injecting high QC samples. Overall, the evaluation shows no residue in the blank samples. The RSB blank residue acceptance criteria for the study sample analysis will be ≤ 5.0% of the average peak area of the unlabeled peptide in the QC0 (endogenous) samples.

[0189] Precision and accuracy

[0190] The precision and accuracy of the HPPD method for commercial crops are evaluated using the QC samples described herein. Three precision and accuracy runs are performed. All runs meet the run acceptance criteria. The precision (CV) and accuracy (bias) data for within-batch and between-batch runs are summarized in Tables 14 to 23 (one table for each plant species / matrix).

[0191] For within-batch and between-batch, the CV of the QC0 samples is less than 25.0%, and the CV of the low, medium, and high QC samples is less than 20.0%. Therefore, the method precision evaluation is appropriate.

[0192] For within-batch and between-batch, the bias of the low, medium, and high QC samples is within ±20.0%; therefore, the method accuracy evaluation is appropriate.

[0193] The longest injection time for precision and accuracy batches is approximately 36 hours and 58 minutes (batch size of 187 injections), so this was determined to be the longest injection time for the assays for the study sample analysis.

[0194] Trend analysis was performed on the QC0 (endogenous) samples from the precision and accuracy runs. The QC0 (endogenous) samples were also analyzed for sample analysis to extend the trend analysis.

[0195] Table 14. Intra- and inter-batch precision and accuracy results for HPPD in barley seeds

[0196]

[0197] Nominal = actual concentration used for calculation

[0198] For each QC in each run, N = 3

[0199] N / Ap = not applicable

[0200] Table 15 Intra- and inter-batch precision and accuracy results for HPPD in maize seeds

[0201]

[0202] Nominal = actual concentration used for calculation

[0203] For each QC in each run, N = 3

[0204] N / Ap = not applicable

[0205] Table 16. Intra- and inter-batch precision and accuracy results for HPPD in rice seeds

[0206]

[0207] Nominal = actual concentration used for calculation

[0208] For each QC in each run, N = 3

[0209] N / Ap = not applicable

[0210] Table 17. Intra- and inter-batch precision and accuracy results for HPPD in soybean seeds

[0211]

[0212] Nominal = actual concentration used for calculation

[0213] For each QC in each run, N = 3

[0214] N / Ap = not applicable

[0215] Table 18. Results of within - batch and between - batch precision and accuracy of HPPD in wheat seeds

[0216]

[0217] Nominal = actual concentration used for calculation

[0218] For each QC in each run, N = 3

[0219] N / Ap = not applicable

[0220] Table 19. Results of within - batch and between - batch precision and accuracy of HPPD in barley feed

[0221]

[0222] Nominal = actual concentration used for calculation

[0223] For each QC in each run, N = 3

[0224] N / Ap = not applicable

[0225] Table 20. Results of within - batch and between - batch precision and accuracy of HPPD in maize feed

[0226]

[0227] Nominal = actual concentration used for calculation

[0228] For each QC in each run, N = 3

[0229] N / Ap = not applicable

[0230] Table 21. Results of within - batch and between - batch precision and accuracy of HPPD in oat feed

[0231]

[0232] Nominal = actual concentration used for calculation

[0233] For each QC in each run, N = 3

[0234] N / Ap = not applicable

[0235] Table 22. Results of within - batch and between - batch precision and accuracy of HPPD in soybean feed

[0236]

[0237] Nominal = actual concentration used for calculation

[0238] For each QC in each run, N = 3

[0239] N / Ap = Not applicable

[0240] Table 23. Intra - and inter - batch precision and accuracy results of HPPD in wheat feed

[0241]

[0242] Nominal = actual concentration used for calculation

[0243] For each QC in each run, N = 3

[0244] N / Ap = Not applicable

[0245] Extraction efficiency

[0246] Table 24 summarizes the efficiency of the method for extracting proteins from commodity crops. All plant species / matrices for HPPD achieved acceptable extraction efficiency in a single iteration. 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%) indicated that the method met the acceptable extraction efficiency assessment. In addition, the CV values of HPPD in all plant species / matrices were below 20.0%, and the final extraction amounts of all commodity crops except barley and wheat seeds were below 5.0%.

[0247] Table 24. Extraction efficiency of HPPD from commodity crops

[0248]

[0249]

[0250] N = 4 extraction iterations and 3 replicates per iteration

[0251] Extraction efficiency was performed in the 3rd run

[0252] See Appendix A, Table A14 for details

[0253] Italic and bold values exceed the acceptance criteria, see Section 5.1.3

[0254] Table 25 summarizes the dry - extraction stability of alternative peptides at a nominal temperature of - 20 °C for 6 days (146 hours 16 minutes). For the stability assessment of the dried samples, the average peak response ratio of the stability samples ≤ 25.0% CV and the % difference of the stability samples compared to the Day 0 QC samples was within ± 25.0%.

[0255] Table 25. Stability of Dry Extracts of Commodity Crops at a Nominal Temperature of -20 °C for 6 Days (146 Hours 16 Minutes)

[0256]

[0257]

[0258] For all QCs, N = 3; N / A p = not applicable

[0259] a % difference - (QC stability - Day 0 QC) / Day 0 QC x 100

[0260] Dry stability was performed in the 7th run

[0261] Refer to Appendix A, Table A15 for details

[0262] The aim of this study was to validate an MS-based method for the quantification of 4-hydroxyphenylpyruvate dioxygenase (HPPD) protein in various commodity crops (barley, maize, rice, soybean and wheat seeds, and barley, maize, oats, soybean and wheat feeds) using LC-MS / MS. Specific surrogate peptides for the HPPD protein were used to determine the relative concentrations. Commodity crop samples were used to evaluate method performance parameters, including specificity, linearity, limit of quantification, carryover, precision and accuracy, extraction efficiency and stability.

[0263] The evaluation of specificity showed no significant interference in the retention times of the unlabeled peptides (average ratio difference between two transitions of blank and buffer < 30.0%) and SIL peptides (SIL signal of QC0 (endogenous) ≤ 5.0%) in any of the tested commodity crop samples. All specificity parameters met the acceptance criteria.

[0264] The lower limits of quantification and quantification ranges (in fmol / mg DW and μg / g DW, respectively) of HPPD in commodity crops are shown in Table 26.

[0265] Table 26. List of Commodity Crops, Surrogate Peptides, Lower Limits of Quantification and Quantification Ranges

[0266]

[0267] The within-batch and between-batch precision and accuracy of the method are summarized in Table 27.

[0268] Table 27. Within-batch and Between-batch Precision and Accuracy Ranges of HPPD in Commodity Crops

[0269]

[0270] Linear equations were determined to adequately represent the concentration / detector response relationship of HPPD in all commodity crops. A data weight of 1 / x was used for all commodity crops.

[0271] Efficiency of protein extraction methods: 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 for all commodity crops was below 20.0%, except for wheat seeds with a CV of 27.5%. However, the QC0 (endogenous) precision and accuracy results from single extractions of wheat seeds indicated a precision of ≤20.0%.

[0272] Treatment stability (dry extracts) was evaluated at -20°C for 6 days. The stability evaluation met the acceptance criteria. For all commodity crops, CV ≤ 25.0% and the difference % in the peak area ratio between stability and day 0 QC was within ±25.0%.

[0273] All evaluated performance parameters met the specified acceptance criteria, except for the final extraction of 8.2% for barley and 6.2% for wheat seeds and the precision of 27.5% CV for the first round of extraction of wheat seeds. Since the concentration of HPPD in sample analysis will be determined by single extraction (first round) and adjusted according to the extraction efficiency %, and the precision of wheat seeds in the precision and accuracy runs reached ≤20.0%, there was no impact on the results of the study. Based on the results of this study, the mass spectrometry-based method has been demonstrated to be suitable for quantifying HPPD protein in the list of commodity crops evaluated in this study according to GLPS.

[0274] Although the invention has been described in connection with specific embodiments thereof, it is to be understood that the apparatus of the invention is capable of further modification. This patent application is intended to cover any variations, uses, or adaptations of the invention in general that follow the principles of the invention and include such departures from the disclosure as come within known or customary practice in the art to which the invention pertains and as may be applied to the key features set forth above and fall within the scope of the appended claims.

[0275] All publications and patent applications mentioned in this specification indicate the level of skill of those skilled in the art to which the invention pertains. All publications and patent applications are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Sequence Listing <110> Syngenta Crop Protection AG S. Yang <120> Compositions and methods for protein detection <130> 81875-WO-REG-ORG-P1 <150> 62 / 839988 <151> April 29, 2019 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> soybean <400> 1 Gly Asn Phe Ser Glu Leu Phe Lys 1 5 <210> 2 <211> 8 <212> PRT <213> maize <400> 2 Gly Asn Phe Ser Gln Leu Phe Lys 1 5

Claims

1. A labeled surrogate peptide that functions in mass spectrometry to selectively detect or quantify 4-hydroxyphenylpyruvate dioxygenase (HPPD) protein in a protein mixture in one or more biological samples from one or more crop plants, said surrogate peptide comprising a label and consisting of an amino acid sequence selected from GNFSELFK (SEQ ID NO:1) and GNFSQLFK (SEQ ID NO:2).

2. The labeled surrogate peptide according to claim 1, wherein the peptide is labeled by incorporation of stable isotope-labeled (SIL) amino acids.

3. The labeled surrogate peptide according to claim 2, wherein the SIL amino acid is lysine.

4. The labeled surrogate peptide according to claim 1, wherein the plant is barley, rice, soybean, wheat, oats or maize.

5. The surrogate peptide according to claim 4, wherein the plant is barley, soybean, wheat or rice, and the surrogate peptide comprises a label and consists of the amino acid sequence of SEQ ID NO:

1.

6. The surrogate peptide according to claim 4, wherein the plant is maize, and the surrogate peptide comprises a label and consists of the amino acid sequence of SEQ ID NO:

2.

7. A kit comprising: (i) a first synthetic labeled surrogate peptide that comprises a label and consists of the amino acid sequence of SEQ ID NO:1; and (ii) a second synthetic labeled surrogate peptide that comprises a label and consists of the amino acid sequence of SEQ ID NO:

2.

8. A method for simultaneously detecting or quantifying one or more target 4-hydroxyphenylpyruvate dioxygenase (HPPD) proteins in a complex biological sample from a crop plant comprising a mixture of the one or more target HPPD proteins and non-target proteins, the method comprising: a. obtaining a biological sample from the crop plant; b. extracting proteins from the biological sample to obtain an extract comprising a protein mixture; c. reducing the amount of insoluble proteins in the extract of step b to obtain a concentrated extract of soluble proteins; d. digesting the soluble proteins in the extract of step c to obtain an extract comprising peptide fragments, wherein the peptide fragments comprise at least one unlabeled surrogate peptide specific for one of the one or more target HPPD proteins; e. concentrating the peptide fragments in the extract of step d; f. adding one or more synthetic labeled surrogate peptides that comprise a label and consist of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, wherein each synthetic labeled surrogate peptide in the mixture is an analog of an unlabeled surrogate peptide of the target HPPD protein and has the same amino acid sequence as the unlabeled surrogate peptide of the target HPPD protein, and wherein the number of labeled surrogate peptides added is equal to the number of target HPPD proteins in the mixture; g. Concentrating the unlabeled surrogate peptide and the synthetically labeled surrogate peptide by reducing the amount of non-surrogate peptide in the mixture; h. Decomposing the peptide fragment mixture from step g by liquid chromatography; i. Analyzing the peptide fragment mixture obtained from step h by mass spectrometry, wherein the detection of the transition ion fragment of the synthetically labeled surrogate peptide indicates the presence of the target HPPD protein that derivatizes the unlabeled surrogate peptide; and optionally, j. Calculating the amount of the target HPPD protein in the biological sample by comparing the mass spectrometry signal generated by the transition ion fragment of step i with the mass spectrometry signal generated by the transition ion of the synthetically labeled surrogate peptide of the target HPPD protein.

9. The method according to claim 8, wherein the crop plant is barley, soybean, wheat or rice, and the synthetically labeled surrogate peptide consists of the amino acid sequence of SEQ ID NO:

1.

10. The method according to claim 8, wherein the crop plant is maize, and the synthetically labeled surrogate peptide consists of the amino acid sequence of SEQ ID NO:

2.

11. The method according to claim 9 or 10, wherein the synthetically labeled surrogate peptide is labeled by incorporating stable isotope-labeled (SIL) amino acids.

12. The method according to claim 11, wherein the SIL amino acid is lysine.

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