WO2016025516A1

WO2016025516A1 - Methods and systems for selective quantitation and detection of allergens

Info

Publication number: WO2016025516A1
Application number: PCT/US2015/044710
Authority: WO
Inventors: Trent James OMAN; Barry William SCHAFER; Ryan Christopher HILL; Guomin Shan
Original assignee: Dow Agrosciences Llc
Priority date: 2014-08-11
Filing date: 2015-08-11
Publication date: 2016-02-18
Also published as: CA2958063A1; EP3180622A4; CN106796242A; AU2015301806A1; BR112017002622A2; EP3180622A1

Abstract

The invention relates to methods and systems taking advantage of bioinformatic investigations to identify candidate signature peptides for quantitative multiplex analysis of complex protein samples from plants, plant parts, and/or food products using mass spectroscopy. Provided are use and methods for selecting candidate signature peptides for quantitation using a bioinformatic approach. Also provided are systems comprising a chromatography and mass spectrometry for using selected signature peptides.

Description

METHODS AND SYSTEMS FOR SELECTIVE QUANTITATION

AND DETECTION OF ALLERGENS

PRIORITY CLAIM

This application claims the benefit of the filing date of United States Provisional

Patent Applications: Serial No. 62/035,744, filed August 1 1 , 2014, for "Methods and Systems for Selective Quantitation and Detection of Allergens Including GLY M 1"; Serial No. 62/035,731 , filed August 1 1, 2014, for "Methods and Systems for Selective Quantitation and Detection of Allergens Including GLY M 3"; Serial No. 62/035,768, filed August 1 1 , 2014, for "Methods and Systems for Selective Quantitation and Detection of Allergens Including GLY M 4"; Serial No. 62/035,800, filed August 1 1 , 2014, for "Methods and Systems for Selective Quantitation and Detection of Allergens Including GLY M 5"; Serial No. 62/035,858, filed August 1 1 , 2014, for "Methods and Systems for Selective Quantitation and Detection of Allergens Including GLY M 6 Gl SUBUNIT"; Serial No. 62/035,876, filed August 1 1 , 2014, for "Methods and Systems for Selective Quantitation and Detection of Allergens Including GLY M 6 G2 SUBUNIT"; Serial No. 62/035,920, filed August 1 1 , 2014, for "Methods and Systems for Selective Quantitation and Detection of Allergens Including GLY M 6 G3 SUBUNIT"; Serial No. 62/036,926, filed August 13, 2014, for "Methods and Systems for Selective Quantitation and Detection of Allergens Including GLY M 6 G4 SUBUNIT"; and Serial No. 62/035,944, filed August 1 1 , 2014, for "Methods and Systems for Selective Quantitation and Detection of Allergens Including GLY M 6 Precursor," the disclosure of each of which is hereby incorporated herein in its entirety by this reference.

BACKGROUND

The current methods for analysis of gene expression in plants that are preferred in the art include DNA-based techniques (for example PCR and/or RT-PCR); the use of reporter genes; Southern blotting; and immunochemistry. All of these methodologies suffer from various shortcomings. Detection of known and potential allergens in plants, plant parts, and/or food products is an important subject for public safety.

Although mass spectrometry has been disclosed previously, existing approaches are lim ited without selected and sensitive quantitation. There remains a need for a high- throughput method for selected and sensitive quantitation of known and/or potential allergens in plant, plant parts, and/or food products.

DISCLOSURE

The invention relates to methods and systems taking advantage of bioinformatic investigations to identify candidate signature peptides for quantitative multiplex analysis of complex protein samples from plants, plant parts, and/or food products using mass spectrometry. Provided are use and methods for selecting candidate signature peptides for quantitation using a bioinformatic approach. Also provided are systems comprising a chromatography and mass spectrometry for using selected signature peptides.

In one aspect, provided is a method of selecting candidate signature peptide for quantitation of known allergen and potential allergens from a plant-based sample. The method comprises:

(a) identifying potential allergens based on homology to at least one known allergen protein sequence;

(b) performing sequence alignment of the at least one known allergen and potential allergens identified in step (a);

(c) selecting a consensus sequence or representative sequence based on the sequence alignment;

(d) determining a plural of candidate signature peptides based on conservative regions or domains from the sequence alignment and in silico digestion data of the consensus sequence or representative sequence selected in Step (c); and

(e) quantitating the amount of the at least one known allergen and potential allergens in the plant-based sample based on measurements of the signature peptides.

In one embodiment, the quantitating step uses a column chromatography and mass spectrometry. In another embodiment, the quantitating step comprises measuring the plural of candidate signature peptides using high resolution accurate mass spectrometry (HRAM MS). In another embodiment, the quantitating step comprises calculating corresponding peak heights or peak areas of the candidate signature peptides from mass spectrometry. In another embodiment, the quantitating step comprises comparing data from high fragmentation mode and low fragmentation mode from mass spectrometry. In one embodiment, the at least one known allergen comprises at least one allergen selected from the group consisting of Gly m 1, Gly m 3, Gly m 4, Gly m 5 (beta- conglycinin), Gly m 6 (Glycinin) Gl , Gly m 6 (Glycinin) G2, Gly m 6 (Glycinin) G3, Gly m 6 (Glycinin) G4, Gly m 6 (Glycinin) precursor, Kunitz trypsin inhibitor 1 , Kunitz trypsin inhibitor 3, Gly m Bd 28 K, Gly m Bd 30 K, Gly m 8 (2S albumin), Lectin, and lipoxygenase. In another embodiment, the at least one known allergen comprises Gly m 1, Gly m 3, Gly m 4, Gly m 5 (beta-conglycinin), Gly m 6 (Glycinin) Gl, Gly m 6 (Glycinin) G2, Gly m 6 (Glycinin) G3, Gly m 6 (Glycinin) G4, or Gly m 6 (Glycinin) precursor.

In another embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 12, 28, 29, 30, 31 , 32, 33, 34, or 35 for Gly m 1. In a further embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 12, 28, or 29 for Gly m l . In another embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 13, 38, 39, or 40 for Gly m 3. In a further embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 13 or 38 for Gly m 3. In another embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 14, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 for Gly m 4. In a further embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 14 or 42 for Gly m 4. In another embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 15, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, or 74 for Gly m 5. In a further embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 15 or 61 for Gly m 5. In another embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 16 or 95 for Gly m 6 Gl . In another embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 17 or 107 for Gly m 6 G2. In another embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 18 for Gly m 6 G3. In another embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 19, 130, 131 , 132, 133, or 134 for Gly m 6 G4. In a further embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 19 or 130 for Gly m 6 G4. In another embodiment, the consensus sequence or representative sequence of step (c) comprises SEQ ID NO: 20 for Gly m 6 precursor.

In one embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 12 and 30-35 for Gly m 1 . In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 13 and 39-40 for Gly m 3. In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 14 and 43-51 for Gly m 4. In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 15 and 62- 74 for Gly m 5. In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 16 and 95 for Gly m 6 Gl . In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 17 and 107 for Gly m 6 G2. In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 19 and 131-134 for Gly m 6 G4.

In another embodiment, the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 1, 36, and 37 for Gly m 1. In another embodiment, the candidate signature peptides comprise SEQ ID NOs: 36 and 37 for Gly m 1. In another embodiment, the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 2 and 41 for Gly m 3. In another embodiment, the candidate signature peptides comprise SEQ ID NO: 41 for Gly m 3. In another embodiment, the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 52-57 for Gly m 4. In another embodiment, the candidate signature peptides comprise SEQ ID NOs: 52-57 for Gly m 4. In another embodiment, the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 3 and 75-94 for Gly m 5. In another embodiment, the candidate signature peptides comprise SEQ ID NOs: 75-94 for Gly m 5. In another embodiment, the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 96-106 for Gly m 6 Gl . In another embodiment, the candidate signature peptides comprise SEQ ID NOs: 96-106 for Gly m 6 Gl . In another embodiment, the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 4 and 108-120 for Gly m 6 G2. In another embodiment, the candidate signature peptides comprise SEQ ID NOs: 108-120 for Gly m 6 G2. In another embodiment, the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 5 and 121 -129 for Gly m 6 G3. In another embodiment, the candidate signature peptides comprise SEQ ID NOs: 121 -129 for Gly m 6 G3. In another embodiment, the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 135- 1 56 and 58 for Gly m 6 G4. In another embodiment, the candidate signature peptides comprise SEQ ID NOs: 135-156 and 58 for Gly m 6 G4. In another embodiment, the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 6, 59, and 60 for Gly m 6 precursor. In another embodiment, the candidate signature peptides comprise SEQ ID NOs: 59 and 60 for Gly m 6 precursor. In another embodiment, the plant-based sample comprises a soybean seed or part of a soybean seed.

In another aspect, provided is a system for quantitating one or more protein of interest with known amino acid sequence in a plant-based sample. The system comprises:

(a) a high-throughput means for extracting proteins from a plant-based sample;

(b) a process module for digesting extracted proteins with at least one protease;

(c) a separation module for separating peptides in a single step;

(d) a selection module for selecting a plural of signature peptides for at least one known allergen and potential allergens; and

(e) a mass spectrometry for measuring the plural of signature peptides.

In one embodiment, the separation module comprises a column chromatography. In a further embodiment, the column chromatography comprises a liquid column chromatography. In another embodiment, the mass spectrometry comprises a high resolution accurate mass spectrometry (HRAM MS). In another embodiment, the selection module uses a method provided herein.

In one embodiment, the one or more protein of interest with known amino acid sequence in a plant-based sample comprises potential allergens. In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 12 and 30-35 for Gly m l . In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 13 and 39-40 for Gly m 3. In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 14 and 43-51 for Gly m 4. In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 15 and 62-74 for Gly m 5. In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 16 and 95 for Gly m 6 G l . In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 1 7 and 107 for Gly m 6 G2. In another embodiment, the potential allergens comprise at least one sequence selected from SEQ ID NOs: 1 and 131 -134 for Gly m 6 G4.

In another embodiment, the signature peptides comprise at least one sequence selected from SEQ ID NOs: 1 , 36, and 37 for Gly m 1 . In another embodiment, the signature peptides comprise SEQ ID NOs: 36 and 37 for Gly m 1 . In another embodiment, the signature peptides comprise at least one sequence selected from SEQ ID NOs: 2 and 41 for Gly m 3. In another embodiment, the signature peptides comprise SEQ ID NO: 41 for Gly m 3. In another embodiment, the signature peptides comprise at least one sequence selected from SEQ ID NOs: 52-57 for Gly m 4. In another embodiment, the signature peptides comprise SEQ ID NOs: 52-57 for Gly m 4. In another embodiment, the signature peptides comprise at least one sequence selected from SEQ ID NOs: 3 and 75-94 for Gly m 5. In another embodiment, the signature peptides comprise SEQ ID NOs: 75-94 for Gly m 5. In another embodiment, the signature peptides comprise at least one sequence selected from SEQ ID NOs: 96-106 for Gly m 6 Gl . In another embodiment, the signature peptides comprise SEQ ID NOs: 96-106 for Gly m 6 Gl . In another embodiment, the signature peptides comprise at least one sequence selected from SEQ ID NOs: 4 and 108-120 for Gly m 6 G2. In another embodiment, the signature peptides comprise SEQ ID NOs: 108-120 for Gly m 6 G2. In another embodiment, the signature peptides comprise at least one sequence selected from SEQ ID NOs: 5 and 121 -129 for Gly m 6 G3. In another embodiment, the signature peptides comprise SEQ ID NOs: 121-129 for Gly m 6 G3. In another embodiment, the signature peptides comprise at least one sequence selected from SEQ ID NOs: 135-156 and 58 for Gly m 6 G4. In another embodiment, the signature peptides comprise SEQ ID NOs: 135-156 and 58 for Gly m 6 G4. In another embodiment, the signature peptides comprise at least one sequence selected from SEQ ID NOs: 6, 59, and 60 for Gly m 6 precursor. In another embodiment, the signature peptides comprise SEQ ID NOs: 59 and 60 for Gly m 6 precursor. In another embodiment, the plant-based sample comprises a soybean seed or part of a soybean seed.

In another aspect, provided is a high-throughput method of quantitating at least one allergen with known amino acid sequence and homologous potential allergens in a plant- based sample. The method comprises using the system provided herein.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows a representative analysis work flow for the methods and systems disclosed herein.

FIGs. 2A - 2C show representative SRM LC-MS/MS for selected signature peptides SEQ ID NO: 1 SYPSNATCPR; SEQ ID NO: 36 ALGILNLNR; and SEQ ID NO: 37 NLQL1LNSCGR from trypsin digested soybean sample chromatogram for Gly m 1. FIG. 2D shows representative SRM LC-MS/MS Standard Chromatogram - 500 ng/mL Synthetic Peptide SEQ ID NO: 1 SYPSNATCPR natural abundance peptide and heavy isotope labeled peptide transitions.

FIG. 2E shows sequences alignments among Gly m 1 and potential homologs of Gly m l .

FIG. 3A shows representative SRM LC-MS/MS for selected signature peptide SEQ ID NO: 2 YMVIQGEPGAVIR from trypsin digested soybean sample chromatogram for Gly m 3.

FIG. 3B shows representative SRM LC-MS/MS Standard Chromatogram - 500 ng/mL Synthetic Peptide SEQ ID NO: 2 YMVIQGEPGAVIR natural abundance peptide and heavy isotope labeled peptide transitions.

FIG. 3C shows sequences alignments among Gly m 3 and potential homologs of Gly m 3.

FIGs. 4A - 4F show representative SRM LC-MS/MS for selected signature peptides SEQ ID NO: 52 MGVFTFEDEINSPVAPATLYK; SEQ ID NO: 53 ALDSFK; SEQ ID NO: 54 SVENVEGNGGPGTIK; SEQ ID NO: 55 ITFLEDGETK; SEQ ID NO: 56 FVLHK; and SEQ ID NO: 57 AIEAYLLAHPDYN from trypsin digested soybean sample chromatogram for Gly m 4.

FIG. 4G shows sequences alignments among Gly m 4 and potential homologs of Gly m 4.

FIGs. 5A - 5U show representative SRM LC-MS/MS for selected signature peptides SEQ ID NO: 3 NILEASYDTK; SEQ ID NO: 75 CNLLK; SEQ ID NO: 76 EEDEDEQPRPIPFPRPQPR; SEQ ID NO: 77 EEQEWPR; SEQ ID NO: 78 QFPFPRPPHQK; SEQ ID NO: 79 ESEESEDSELR; SEQ ID NO: 80 NPFLFGSNR; SEQ ID NO: 81 FETLFK; SEQ ID NO: 82 SPQLQNLR; SEQ ID NO: 83 LQSGDALR; SEQ ID NO: 84 VPSGTTYYVVNPDNNENLR; SEQ ID NO: 85 FESFFLSSTEAQQSYLQGFSR; SEQ ID NO: 86 FEEINK; SEQ ID NO: 87 VLFSR; SEQ ID NO: 88 TISSEDKPFNLR; SEQ ID NO: 89 DP1YSNK; SEQ ID NO: 90 FFEITPEK; SEQ ID NO: 91 A I VILVINEGDANIELVGLK; SEQ ID NO: 92 EQQQEQQQEEQPLEVR; SEQ ID NO: 93

NFLAGSQDNVISQ1PSQVQELAFPGSAQAVEK; and SEQ ID NO: 94 ESYFVDAQPK from trypsin digested soybean sample chromatogram for Gly m 5

FIG. 5V shows representative SRM LC-MS/MS Standard Chromatogram - 500 ng/mL Synthetic Peptide SEQ ID NO: 3 NILEASYDTK natural abundance peptide and heavy isotope labeled peptide transitions.

FIG. 5W show sequences alignments among Gly m 5 and potential homologs of Gly m 5.

FIG. 6A - 6K show representative SRM LC -MS/MS for selected signature peptides

SEQ ID NO: 96 LVFSLCFLLFSGCCFAFSSR; SEQ ID NO: 97 EQPQQNECQIQK; SEQ ID NO: 98 RP S YTNGPQEI YIQQGK; SEQ ID NO: 99 HQQEEENEGGSILSGFTLEFLEHAFSVDK; SEQ ID NO: 100 HCQRPR; SEQ ID NO: 101 HNIGQTSSPDIYNPQAGSVTTATSLDFPALSWLR; SEQ ID NO: 102 ALIQVVNCNGER; SEQ ID NO: 103 VFDGELQEGR; SEQ ID NO: 104 TNDTPMIGTLAGANSLLNALPEEVIQHTFNLK; SEQ ID NO: 105 NNNPFK; and SEQ ID NO: 106 FLVPPQESQK from trypsin digested soybean sample chromatogram for Gly m 6 Gl .

FIG. 6L shows a sequence alignment between Gly m 6 Gl and a potential homolog of Gly m 6 Gl .

FIGs. 7A - 7N show representative SRM LC-MS/MS for selected signature peptides SEQ ID NO: 4 VTAPAMR; SEQ ID NO: 108 LVLSLCFLLFSGCFALR; SEQ ID NO: 109 EQAQQNECQIQK; SEQ ID NO: 1 10 RPSYTNGPQEIYIQQGNGIFGMIFPGC PSTYQEPQESQQR; SEQ ID NO: 1 1 1 SQRPQDR; SEQ ID NO: 1 12 QQEEENEGSNILSGFAPEFLK; SEQ ID NO: 1 13 EAFGVNMQIVR; SEQ ID NO: 1 14 KPQQEEDDDDEEEQPQCVETDK; SEQ ID NO: 1 15 LSAQYGSLR; SEQ ID NO: 1 16 NAMFVPHYTLNANSIIYALNGR; SEQ ID NO: 1 17 ALVQVVNCNGER; SEQ ID NO: 1 18 VFDGELQEGGVLIVPQNFAVAAK; SEQ ID NO: 1 19 TNDRPSIGNLAGANSLLNALPEE-VIQHTFNLK; and SEQ ID NO: 120 NNNPFSFLVPPQESQR from trypsin digested soybean sample chromatogram for Gly m 6 G2.

FIG. 70 shows representative SRM LC-MS/MS Standard Chromatogram - 500 ng/mL Synthetic Peptide SEQ ID NO: 4 VTAPAMR natural abundance peptide and heavy isotope labeled peptide transitions.

FIG. 7P shows a sequence alignment between Gly m 6 G2 and a potential homolog of Gly m 6 G2.

FIGs. 8A - 8J show representative SRM LC-MS/MS for selected signature peptides SEQ ID NO: 5 NNNPFSFLVPPK: SEQ ID NO: 121 LVLSLCFLLFSGCCFAFSFR; SEQ ID NO: 122 EQPQQNECQIQR; SEQ ID NO: 123 QQEEENEGGSILSGFAPEFLEHAFVVDR; SEQ ID NO: 124 LQGENEEEEK; SEQ ID NO: 125 GGLSVISPPTEEQQQRPEEEEKPDCDEK; SEQ ID NO: 126 HCQSQSR; SEQ ID NO: 127 LSAQFGSLR; SEQ ID NO: 128 VFDGELQEGQVLIVPQNFAVAAR; and SEQ ID NO: 129 TNDRPSIGNLAGANSLLNALPEEVIQQTFNLR from trypsin digested soybean sample chromatogram for Gly m 6 G3.

FIG. 8K shows representative SRM LC-MS/MS Standard Chromatogram - 500 ng/mL Synthetic Peptide SEQ ID NO: 5 NNNPF SFL VPPK natural abundance peptide and heavy isotope labeled peptide transitions.

FIGs. 9A - 9X show representative SRM LC-MS/MS for selected signature peptides SEQ ID NO: 6 ADFYNPK; SEQ ID NO: 135 LNECQLNNLNALEPDHR; SEQ ID NO: 136 CAGVTVSK; SEQ ID NO: 137 LTLNR; SEQ ID NO: 138 MIIIAQGK; SEQ ID NO: 139 GALGVAIPGCPETFEEPQEQSNR; SEQ ID NO: 140 QQLQDSHQK; SEQ ID NO: 141 VFYLAGNPDIEYPETMQQQQQQK; SEQ ID NO: 142 QGQHQQEEEEEGGSVLSGFSK; SEQ ID NO: 143 HFLAQSFNTNEDIAEK; SEQ ID NO: 144 QIVTVEGGLSVISPK; SEQ ID NO: 145

WQEQQDEDEDEDEDDEDEQIPSHPPR; SEQ ID NO: 146 RPSHGK; SEQ ID NO: 147 EQDEDEDEDEDKPRP SRP SQGK; SEQ ID NO: 148 QEEPR; SEQ ID NO: 149 NGVEENICTLK; SEQ ID NO: 150 LHENIARPSR; SEQ ID NO: 151 ISTLNSLTLPALR; SEQ ID NO: 152 QFQLSAQYVVLYK; SEQ ID NO: 153 NGIYSPHWNLNANSVIYVTR; SEQ ID NO: 154 DVFR; SEQ ID NO: 155 AIPSEVLAHSYNLR; SEQ ID NO: 156 QSQVSELK; and SEQ ID NO: 58 YEGNWGPLVNPESQQGSPR from trypsin digested soybean sample chromatogram for Gly m 6 G4.

FIG 9Y shows sequences alignments among Gly m 6 G4 and potential homologs of

Gly m 6 G4.

FIGs. 10A, 10B, AND I OC show representative SRM LC-MS/MS for selected signature peptides SEQ ID NO: 6 ADFYNPK, SEQ ID NO: 59 GALQCKPGCPETFEEPQEQSNR, and SEQ ID NO: 60 LQSPDDER from trypsin digested soybean sample chromatogram for Gly m 6 precursor.

MODE(S) FOR CARRYING OUT THE INVENTION It is of significance to enable a sensitive multiplex assay that is capable of selectively detecting and measuring levels of proteins of interest. Currently, relevant technologies for protein expression detection rely heavily on traditional immunochemistry technologies which present a challenge to accommodate the volume of data required to generate per sample.

Soybean is a multi-billion dollar commodity due to its balanced composition of

2:2: 1 protein, starch, and oil by weight. Many seeds, including soybeans, contain proteins that are allergens and anti-nutritional factors. As such, there are concerns regarding the potential of altering allergen levels in genetically-modified soybean varieties when compared to varieties developed through traditional breeding. The measurement of allergen levels in crops has been achieved almost exclusively by immunoassays, such as enzyme-linked immunosorbent assays (ELISA) or IgE-immunoblotting; however, these methods suffer from limited sensitivity and specificity and high variability.

There has been recent interest in developing LC-MS/MS based methods to quantify several plant-expressed proteins in a single analysis. Analysis using these "signature peptides" involves tracking protein expression levels by quantifying several highly specific digest fragments of the proteins of interest. This can be typically accomplished using liquid chromatography coupled with selected reaction monitoring (SRM) tandem mass spectrometry. Improved multiplexed LC-MS/MS methods and systems are provided herein to enable simultaneous quantitation(s) of several allergen proteins in transgenic and non- transgenic soybean. Methods and systems provided herein are validated for analytical figures of merit including accuracy, precision, linearity, limits of detection and quantitation; and for other considerations including sample throughput, transferability, and ease of use. The allergens can be quantified using a multiplexing format and samples can be harvested from the field, processed, and analyzed/quantitated for example within a day (twenty-four hours) window (from field to measured numerical value). In addition, sample preparations of the methods and systems provided can be fully scalable for high- throughput, thus enabling hundreds of samples to be analyzed in a single batch.

Representative soybean allergens include, for example, Gly m 1 , Gly m 3, Gly m 4, Gly m 5 (beta-conglycinin), Gly m 6 (Glycinin) Gl , Gly m 6 (Glycinin) G2, Gly m 6 (G lycinin) G3, Gly m 6 (Glycinin) G4, Gly m 6 (Glycinin) precursor, Gly m 6 (Glycinin) G4 precursor, Kimitz trypsin inhibitor 1 , Kunitz trypsin inhibitor 3, Gly m Bd 28 K, Gly m Bd 30 K, Gly m 8 (2S albumin), Lectin, and lipoxygenase.

Representative wheat allergens include, for example, profilin (Tri a ! 2), wheat lipid transfer protein 1 (Tri al4), agglutinin isolectin 1 (Tri al 8), omega-5 gliadin - seed storage protein (Tri al9), gliadin (Tri a20; NCBI Accession Nos. M10092, Ml 1073, Ml 1074, Ml 1075, Ml 1076, K03074, and K03075), thioredoxin (Tri a25), high molecular weight glutenin (Tri a26), low molecular weight glutenin (Tri a36), and alpha purothionin (Tri a37).

Representative corn allergens include, for example, maize lipid transfer protein (LTP) (Zea ml 4) and thioredoxin (Zea m25).

Representative corn allergens include, for example, rice profilin A (Ory si 2).

In some embodiments, the methods and systems provided use liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to detect protein expression levels of sixteen different allergens from soybean. In some embodiments, the methods and systems enable analysis of each allergen by itself or combined with additional proteins for a multiplexing assay for qualitative and quantitative analysis in plant matrices.

In some embodiments, the mass spectrometry detection for quantitative studies may be accomplished using selected reaction monitoring, performed on a triple quadrupole mass spectrometer. Using this type of instrumentation, initial mass-selection of ion (peptide) of interest formed in the source, followed by, dissociation of this precursor ion in the collision region of the MS, then mass-selection, and counting, of a specific product (daughter) ion. In some embodiments, the mass spectrometry detection for quantitative studies may be accomplished using selected reaction monitoring (SRM). Using particular type of instrumentation, initial mass-selection of ion of interest formed in the source, followed by, dissociation of this precursor (protein) ion in the collision region of the mass spectrometer (MS), then mass-selection, and counting, of a specific product (peptide) ion. In some embodiment, counts per unit time may provide an integratable peak area from which amounts or concentration of analytes can be determined. In some embodiment, the use of high resolution accurate mass (HRAM) monitoring for quantitation, performed on a HRAM capable mass spectrometer, may include, but is not limited to, hybrid quadrupole- time-of-flight, quadrupole-orbitrap, ion trap-orbitrap, or quadrupole-ion-trap-orbitrap (tribrid) mass spectrometers. Using particular type of instrumentation, peptides are not subject to fragmentation conditions, but rather are measured as intact peptides using full scan or targeted scan modes (for example selective ion monitoring mode or SIM). Integratable peak area can be determined by generating an extracted ion chromatogram for each specific analyte and amounts or concentration of analytes can be calculated. The high resolution and accurate mass nature of the data enable highly specific and sensitive ion signals for the analyte (protein and/or peptide) of interest.

Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "bioconfinement" refers to restriction of the movement of genetically modified plants or their genetic material to designated areas. The term includes physical, physicochemical, biological confinement, as well as other forms of confinement that prevent the survival, spread or reproduction of a genetically modified plants in the natural environment or in artificial growth conditions.

As used herein, the term "complex protein sample" is used to distinguish a sample from a purified protein sample. A complex protein sample contains multiple proteins, and may additionally contain other contaminants.

As used herein, the general term "mass spectrometry" or "MS" refers to any suitable mass spectrometry method, device or configuration including, e.g., electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI) MS, MALDI-time of flight (TOF) MS, atmospheric pressure (AP) MALDI MS, vacuum MALDI MS, or combinations thereof. Mass spectrometry devices measure the molecular mass of a molecule (as a function of the molecule's mass-to-charge ratio) by measuring the molecule's flight path through a set of magnetic and electric fields. The mass-to-charge ratio is a physical quantity that is widely used in the electrodynamics of charged particles. The mass-to-charge ratio of a particular peptide can be calculated, a priori, by one of skill in the art. Two particles with different mass-to-charge ratio will not move in the same path in a vacuum when subjected to the same electric and magnetic fields.

Mass spectrometry instruments consist of three modules: an ion source, which splits the sample molecules into ions; a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields; and a detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present. The technique has both qualitative and quantitative applications. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, determining the structure of a compound by observing its fragmentation, and quantifying the amount of a compound in a sample. A detailed overview of mass spectrometry methodologies 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: Wiley, p. 10.21.1- 10.21.27; Paterson and Aebersold (1995) Electrophoresis 16: 1791-1814; Patterson (1998) Protein identification and characterization by mass spectrometry. In: Current Protocols in Molecular Biology, edited by Ausubel, et al. New York: Wiley, p. 10.22.1-10.22.24; and Domon and Aebersold (2006) Science 312(5771 ):212-17.

As the term is used herein, proteins and/or peptides are "multiplexed" when two or more proteins and/or peptides of interest are present in the same sample.

As used herein, a "plant trait" may refer to any single feature or quantifiable measurement of a plant.

As used herein, the phrase "peptide" or peptides" may refer to short polymers formed from the linking, in a defined order, of a-amino acids. Peptides may also be generated by the digestion of polypeptides, for example proteins, with a protease.

As used herein, the phrase "protein" or proteins" may refer to organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids, however in certain organisms the genetic code can include selenocysteine— and in certain archaea- pyrrolysine. The residues in a protein are often observed to be chemically modified by post-translational modification, which can happen either before the protein is used in the cell, or as part of control mechanisms. Protein residues may also be modified by design, according to techniques familiar to those of skill in the art. As used herein, the term "protein" encompasses linear chains comprising naturally occurring amino acids, synthetic amino acids, modified amino acids, or combinations of any or all of the above.

As used herein, the term "single injection" refers to the initial step in the operation of a MS or LC-MS device. When a protein sample is introduced into the device in a single injection, the entire sample is introduced in a single step.

As used herein, the phrase "signature peptide" refers an identifier (short peptide) sequence of a specific protein. Any protein may contain an average of between 10 and 100 signature peptides. Typically signature peptides have at least one of the following criteria: easily detected by mass spectroscopy, predictably and stably eluted from a liquid chromatography (LC) column, enriched by reversed phase high performance liquid chromatography (RP-HPLC), good ionization, good fragmentation, or combinations thereof. A peptide that is readily quantified by mass spectrometry typically has at least one of the following criteria: readily synthesized, ability to be highly purified (>97%), soluble in ≤=20% acetonitrile, low non-specific binding, oxidation resistant, post-synthesis modification resistant, and a hydrophobicity or hydrophobicity index≥ 10 and≤ 40. The hydrophobicity index can be calculated according to Krokhin, Molecular and Cellular Proteomics 3 (2004) 908, which is incorporated by reference. It's known that a peptide having a hydrophobicity index less than 10 or greater than 40 may not be reproducibly resolved or eluted by a RP-HPLC column.

As used herein, the term "stacked" refers to the presence of multiple heterologous polynucleotides incorporated in the genome of a plant.

Tandem mass spectrometry: In tandem mass spectrometry, a parent ion generated from a molecule of interest may be filtered in a mass spectrometry instrument, and the parent ion subsequently fragmented to yield one or more daughter ions that are then analyzed (detected and/or quantified) in a second mass spectrometry procedure. In some embodiments, the use of tandem mass spectrometry is excluded. In these embodiments, tandem mass spectrometry is not used in the methods and systems provided. Thus, neither parent ions nor daughter ions are generated in these embodiments.

As used herein, the term "transgenic plant" includes reference to a plant which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenic plants initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic plant.

Any plants that provide useful plant parts may be treated in the practice of the present invention. Examples include plants that provide flowers, fruits, vegetables, and grains. As used herein, the phrase "plant" includes dicotyledonous plants and monocotyledonous plants. Examples of dicotyledonous plants include tobacco, Arabidopsis, soybean, tomato, papaya, canola, sunflower, cotton, alfalfa, potato, grapevine, pigeon pea, pea, Brassica, chickpea, sugar beet, rapeseed, watermelon, melon, pepper, peanut, pumpkin, radish, spinach, squash, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber, eggplant, and lettuce. Examples of monocotyledonous plants include corn, rice, wheat, sugarcane, barley, rye, sorghum, orchids, bamboo, banana, cattails, lilies, oat, onion, millet, and triticale. Examples of fruit include banana, pineapple, oranges, grapes, grapefruit, watermelon, melon, apples, peaches, pears, kiwifruit, mango, nectarines, guava, persimmon, avocado, lemon, fig, and berries. Examples of flowers include baby's breath, carnation, dahlia, daffodil, geranium, gerbera, lily, orchid, peony, Queen Anne's lace, rose, snapdragon, or other cut-flowers or ornamental flowers, potted- flowers, and flower bulbs.

The specificity allowed in a mass spectrometry approach for identifying a single protein from a complex sample is unique in that only the sequence of the protein of interest is required in order to identify the protein of interest. Compared to other formats of multiplexing, mass spectrometry is unique in being able to exploit the full length of a protein's primary amino acid sequence to target unique identifier-type portions of a protein's primary amino acid sequence to virtually eliminate non-specific detection. In some embodiments of the present invention, a proteolytic fragment or set of proteolytic fragments that uniquely identifies a protein(s) of interest is used to detect the protein(s) of interest in a complex protein sample.

In some embodiments, disclosed methods enable the quantification or determination of ratios of multiple proteins in a complex protein sample by a single mass spectrometry analysis, as opposed to measuring each protein of interest individually multiple times and compiling the individual results into one sample result.

In some embodiments, the present disclosure also provides methods useful for the development and use of transgenic plant technology. Specifically, disclosed methods may be used to maintain the genotype of transgenic plants through successive generations. Also, some embodiments of the methods disclosed herein may be used to provide high- throughput analysis of non-transgenic plants that are at risk of being contaminated with transgenes from neighboring plants, for example, by cross-pollination. By these embodiments, bioconfinement of transgenes may be facilitated and/or accomplished. In other embodiments, methods disclosed herein may be used to screen the results of a plant transformation procedure in a high-throughput manner to identify transformants that exhibit desirable expression characteristics

The mass-to-charge ratio may be determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and m/z. The voltage and amplitude can be selected so that only ions having a particular m/z travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments can act as a "mass filter" and "mass detector" for the ions injected into the instrument.

Collision-induced dissociation ("CID") is often used to generate the daughter ions for further detection. In CID, parent ions gain energy through collisions with an inert gas, such as argon, and subsequently fragmented by a process referred to as "unimolecular decomposition." Sufficient energy must be deposited in the parent ion so that certain bonds within the ion can be broken due to increased energy.

The mass spectrometer typically provides the user with an ion scan; that is, the relative abundance of each m/z over a given range (for example 10 to 1200 amu). The results of an analyte assay, that is, a mass spectrum, can be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion can be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, molecular standards (e.g., internal standards and external standards) can be run with the samples and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion can be converted into an absolute amount of the original molecule. Numerous other methods for relating the presence or amount of an ion to the presence or amount of the original molecule are well known to those of ordinary skill in the art.

The choice of ionization method can be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc. Ions can be produced using a variety of methods including, but not limited to, electron ionization, chemical ionization, fast atom bombardment, field desorption, and matrix- assisted laser desorption ionization (MALDl), surface enhanced laser desorption ionization (SELDI), desorption electrospray ionization (DESI), photon ionization, electrospray ionization, and inductively coupled plasma. Electrospray ionization refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube, is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber which is heated to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

The effluent of an LC may be injected directly and automatically (i.e., "in-line") into the electrospray device. In some embodiments, proteins contained in an LC effluent are first ionized by electrospray into a parent ion.

Various different mass analyzers can be used in liquid chromatography - mass spectrometry combination (LC-MS). Exemplary mass analyzers include, but not limited to, single quadrupole, triple quadrupole, ion trap, TOF (time of flight), and quadrupole- time of flight (Q-TOF).

The quadrupole mass analyzer may consist of 4 circular rods, set parallel to each other. In a quadrupole mass spectrometer (QMS), the quadrupole is the component of the instrument responsible for filtering sample ions, based on their mass-to-charge ratio (m/z). Ions are separated in a quadrupole based on the stability of their trajectories in the oscillating electric fields that are applied to the rods.

An ion trap is a combination of electric or magnetic fields that captures ions in a region of a vacuum system or tube. Ion traps can be used in mass spectrometry while the ion's quantum state is manipulated.

Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined via a time measurement. Ions are accelerated by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will depend on the mass-to-charge ratio of the particle (heavier particles reach lower speeds). From this time and the known experimental parameters one can find the mass-to-charge ratio of the ion. In some embodiments, the particular instrument used by the methods and/or systems provided may comprise a high fragmentation mode and a low fragmentation mode (or alternatively a non-fragmentation mode). Such different modes may include alternating scan high and low energy acquisition methodology to generate high resolution mass data. In some embodiments, the high resolution mass data may comprise a product data set (for example data derived from product ion (fragmented ions) under the high fragmentation mode) and a precursor data set (for example data derived from precursor ions (unfragmented ions) under the low fragmentation or non-fragmentation mode).

In some embodiments, the methods and/or systems provided use a mass spectrometer comprising a filtering device that may be used in the selection step, a fragmentation device that may be used in the fragmentation step, and/or one or more mass analyzers that may be used in the acquisition and/or mass spectrum creation step or steps.

The filtering device and/or mass analyzer may comprise a quadrupole. The selection step and/or acquisition step and/or mass spectrum creation step or steps may involve the use of a resolving quadrupole. Additionally or alternatively, the filtering device may comprise a two-dimensional or three-dimensional ion trap or time-of-flight (ToF) mass analyzer. The mass analyzer or mass analyzers may comprise or further comprise one or more of a time-of-flight mass analyzer and/or an ion cyclotron resonance mass analyzer and/or an orbitrap mass analyzer and/or a two-dimensional or three-dimensional ion trap.

Filtering by means of selection based upon mass-to-charge ratio (m/z) can be achieved by using a mass analyzer which can select ions based upon m/z, for example a quadrupole; or to transmit a wide m/z range, separate ions according to their m/z, and then select the ions of interest by means of their m/z value. An example of the latter would be a time-of-flight mass analyzer combined with a timed ion selector(s). The methods and/or systems provided may comprise isolating and/or separating the one or more proteins of interest, for example from two or more of a plurality of proteins, using a chromatographic technique for example liquid chromatography (LC). The method may further comprise measuring an elution time for the protein of interest and/or comparing the measured elution time with an expected elution time.

Additionally or alternatively, the proteins of interest may be separated using an ion mobility technique, which may be carried out using an ion mobility cell. Additionally, the proteins of interest may be selected by order or time of ion mobility drift. The method may further comprise measuring a drift time for the proteins of interest and/or comparing the measured drift time with an expected drift time.

In some embodiments, the methods and/or systems provided are label-free, where quantitation can be achieved by comparison of the peak intensity, or area under the mass spectral peak for the precursor or product m/z values of interest between injections and across samples. In some embodiments, internal standard normalization may be used to account for any known associated analytical error. Another label-free method of quantification, spectral counting, involves summing the number of fragment ion spectra, or scans, that are acquired for each given peptide, in a non-redundant or redundant fashion. The associated peptide mass spectra for each protein are then summed, providing a measure of the number of scans per protein with this being proportional to its abundance. Comparison can then be made between samples/injections.

In some embodiments, the ion source is selected from the group consisting of: (1 ) an electrospray ionization ("ESI") ion source; (2) an atmospheric pressure photo ionization ("APPI") ion source; (3) an atmospheric pressure chemical ionization ("APCI") ion source; (4) a matrix assisted laser desorption ionization ("MALDI") ion source; (5) a laser desorption ionization ("LDI") ion source; (6) an atmospheric pressure ionization ("API") ion source; (7) a desorption ionization on silicon ("DIOS") ion source; (8) an electron impact ("El") ion source; (9) a chemical ionization ("CI") ion source; (10) a field ionization ("Fl") ion source; (1 1) a field desorption ("FD") ion source; (12) an inductively coupled plasma ("ICP") ion source; (13) a fast atom bombardment ("FAB") ion source; (14) a liquid secondary ion mass spectrometry ("LSIMS") ion source; (15) a desorption electrospray ionization ("DESI") ion source; (16) a nickel- 63 radioactive ion source; ( 17) an atmospheric pressure matrix assisted laser desorption ionization ion source; and (18) a thermospray ion source.

In some embodiments, the methods and/or systems provided comprise an apparatus and/or control system configured to execute a computer program element comprising computer readable program code means for causing a processor to execute a procedure to implement the methods.

In some embodiments, the methods and/or systems provided use an alternating low and elevated energy scan function in combination with liquid chromatography separation of a plant extract. A list of information for proteins of interest can be provided including, but is not limited to, m/z of precursor ion, m/z of product ions, retention time, ion mobility drift time and rate of change of mobility. During the course of the LC separation and as the target ions elute into the mass spectrometer (and as either low energy precursor ions, or elevated energy product ions are detected, or the retention time window is activated) the mass analyzer of the methods and/or systems provided may select a narrow m/z range (of a variable and changeable width) to pass ions through to the gas cell. Accordingly, the signal to noise ratio can be enhanced significantly for quantification of proteins of interest.

In some embodiments, at a chromatographic retention time when a targeted protein of interest is about to elute into the mass spectrometer ion source, the mass analyzer of the methods and/or systems provided can select a narrow m/z range (of a variable and changeable width) according to the targeted precursor ion. These selected ions are then transferred to an instrument stage capable of dissociating the ions by means of alternate and repeated switches between a high fragmentation mode where the sample precursor ions are substantially fragmented into product ions and a low fragmentation mode (or non- fragmentation mode) where the sample precursor ions are not substantially fragmented. Typically high resolution, accurate mass spectra are acquired in both modes and at the end of the experiment associated precursor and product ions are recognized by the closeness in fit of their chromatographic elution times and optionally other physicochemical properties. The signal intensity of either the precursor ion or the product ion associated with targeted proteins of interest can be used to determine the quantity of the proteins in the plant extract.

Those skilled in the art would understand certain variation can exist based on the disclosure provided. Thus, the following examples are given for the purpose of illustrating the invention and shall not be construed as being a limitation on the scope of the invention or claims. EXAMPLES

Example 1

The methods and systems provided are used for determination of endogenous soybean allergen proteins in soybean seed including Gly m 1 , Gly m 3, Gly m 4, Gly m 5 (beta-conglycinin), Gly m 6, Kunitz trypsin inhibitor 1 , Kunitz trypsin inhibitor 3, Gly m Bd 28 K, Gly m Bd 30 K, and Gly m 8 (2S albumin). A 100 ± 0.5 mg ground soybean seed sample is defatted twice with hexanes and dried before extracting with extraction buffer containing 5 M urea, 2 M thiourea, 50 mM Tris pH 8.0 and 65 iiiM DTT. The sample is sonicated in a water bath for thirty minutes, vortexed for one minute, sonicated for another thirty minutes and centrifuged at > 3,000 rpm for ten minutes at 4°C.

The aqueous supernatant is collected and diluted to bring the endogenous soybean allergen protein concentration into the calibration standard range with extraction buffer. The diluted extract is denatured at 95°C for twenty minutes with the additional 1 M Tris pH 8.0, 0.5 M DTT and deionized water followed by refrigeration at 4°C for ten minutes. The denatured extract is incubated overnight (~ 15 hours) at 37°C with 0.5 mg/mL trypsin enzyme. The digestion reaction is quenched with formic acid water (50/50 v/v) and centrifuge at > 3,000 rpm for tern minutes at 4°C. An aliquot of digested extract is transferred to an autosampler vial and analyzed along with calibration standard by liquid chromatography with positive-ion electrospray (ESI) tandem mass spectrometry (LC- MS/MS). Calibration standards of signature peptides are prepared as listed in Table 1.

The limits of detection (LOD) and limits of quantitation (LOQ) for endogenous soybean allergens in this example are set forth in Table 2, where LOD and LOQ represent protein concentration (ng/mg). Table 2. Limits of detection (LOD) and limits of quantitation (LOQ) for endogenous soybean allergens in Example 1 (LOD and LOQ represent protein concentration)

Allergen Signature peptide LOD LOQ

(ng/mg) (ng/mg)

Gly m 1 SYPSNATCPR (SEQ ID NO: 1) 0.23 0.46

Gly m 3 YMVIQGEPGAVIR (SEQ ID NO: 2) 0.20 0.39

Gly m 5 NILEASYDTK (SEQ ID NO: 3) 1.22 2.44

Glycinin G2 VTAPAMPv (SEQ ID NO: 4) 1.46 2.92

Glycinin G3 NNNPF SFLVPPK (SEQ ID NO: 5) 1.58 3.16

Glycinin ADFYNPK (SEQ ID NO: 6) - - precursor

Kunitz trypsin GGGIEVDSTGK (SEQ ID NO: 7) - - inhibitor 1

Kunitz trypsin GIGTLLSSPYR (SEQ ID NO: 8) - - inhibitor 3

Gly m Bd 28 K NKPQFLAGAASLLR (SEQ ID NO: 9) 5.70 1 1.40

Gly m Bd 30 K GVITQVK (SEQ ID NO: 10) 1.15 2.30

Gly m 8 IMENQSEELEEK (SEQ ID NO: 1 1) 0.25 0.50

Concentrations of allergens are calculated from quantitation of signature peptides (for example Analyst Bioanalytical software for LC-MS/MS), and validated by other methods including enzyme-linked immunosorbent assays (ELISA). Calculated concentrations of allergens from different samples are compared using statistical analysis, and results show good consistency among samples.

Example 2

Several homologous protein sequences for Gly m 1 are identified from public databases including NCBI, Phytozome, and UniProt. Identified sequences (SEQ ID NOs: 12 and 29-35) are analyzed using bioinformatics tools to identify sequence homology and shared sequence composition among the available protein sequences (see FIG. 2E). Specifically this involved the use of Vector NTI Align X alignment tool which performs a CLUSTAL W type alignment. From this analysis, a consensus sequence and/or representative sequence can be determined.

Once the consensus sequence and/or representative sequence is chosen or determined, it is digested in silico to generate candidate signature peptide fragments to be detected and measured by LC-MS. According to the unique approaches provided herein, signature peptides are selected based on the degree of conservation among the available protein sequences, such that the selected signature peptide can be used to quantify all or as many protein isoforms as possible among the identified protein sequences found in the public sequence databases. As a result, quantitation of selected signature peptides can not only measure Gly m 1 itself, but also measure potential allergens which are highly homologous to Gly m 1.

Soybean seed samples are ground to a fine powder, defatted twice with hexane, and extracted with suitable assay buffer (for example 5 M urea, 2 M thiourea, 50 mM Tris (pH 8.0), 65 mM DTT). The samples are sonicated in buffer to extract proteins. The extracted proteins are diluted, denatured, and then proteolytically digested by adding trypsin protease and incubating at 37°C for 15-20 hours. The digestion reactions are acidified with formic acid (pH = 1-2) and are analyzed using LC-MS/MS.

The selected signature peptides can be used for both qualitative and quantitative analysis of Gly m 1 , either by itself or in combination with additional proteins in a multiplexing assay format. In this example, three signature peptides are selected from all peptide possibilities (SEQ ID NO: 1 SYPSNATCPR; SEQ ID NO: 36 ALGILNLNR; and SEQ ID NO: 37 NLQLILNSCGR), and representative quantitation of these signature peptides are shown in FIGs. 2A - 2C. A peptide standard is synthesized for SEQ ID NO: 1 SYPSNATCPR for quantitative and qualitative analyses (see FIG. 2D). Synthetic peptides can directly serve as an analytical reference standard for protein quantitation.

Example 3

Several homologous protein sequences for Gly m 3 are identified from public databases including NCBI, Phytozome, and UniProt. Identified sequences (SEQ ID NOs: 13, 39, and 40) are analyzed using bioinformatics tools to identify sequence homology and shared sequence composition among the available protein sequences (see FIG. 3C). Specifically this involved the use of Vector NTI Align X alignment tool which performs a CLUSTAL W type alignment. From this analysis, a consensus sequence and/or representative sequence can be determined.

Once the consensus sequence and/or representative sequence is chosen or determined, it is digested in silico to generate candidate signature peptide fragments to be detected and measured by LC-MS. According to the unique approaches provided herein, signature peptides are selected based on the degree of conservation among the available protein sequences, such that the selected signature peptide can be used to quantify all or as many protein isoforms as possible among the identified protein sequences found in the public sequence databases. As a result, quantitation of selected signature peptides can not only measure Gly m 3 itself, but also measure potential allergens which are highly homologous to Gly m 3.

The selected signature peptides can be used for both qualitative and quantitative analysis of Gly m 3, either by itself or in combination with additional proteins in a multiplexing assay format. In this example, two signature peptides are selected from all peptide possibilities (SEQ ID NO: 2 YMVIQGEPGAVIR; and SEQ ID NO: 41 GPGGVTVK), and representative quantitation of SEQ ID NO: 2 YMVIQGEPGAVIR is shown in FIG. 3A. A peptide standard is synthesized for SEQ ID NO: 2 YMVIQGEPGAVIR for quantitative and qualitative analyses (see FIG. 3B). Synthetic peptides can directly serve as an analytical reference standard for protein quantitation.

Example 4

Several homologous protein sequences for Gly m 4 are identified from public databases including NCBI, Phytozome, and UniProt. Identified sequences (SEQ ID NOs: 14 and 43-51 ) are analyzed using bioinformatics tools to identify sequence homology and shared sequence composition among the available protein sequences (see FIG. 4G). Specifically this involved the use of Vector NTI Align X alignment tool which performs a CLUSTAL W type alignment. From this analysis, a consensus sequence and/or representative sequence can be determined.

Once the consensus sequence and/or representative sequence is chosen or determined, it is digested in silico to generate candidate signature peptide fragments to be detected and measured by LC-MS. According to the unique approaches provided herein, signature peptides are selected based on the degree of conservation among the available protein sequences, such that the selected signature peptide can be used to quantify all or as many protein isoforms as possible among the identified protein sequences found in the public sequence databases. As a result, quantitation of selected signature peptides can not only measure Gly m 4 itself, but also measure potential allergens which are highly homologous to Gly m 4.

Soybean seed samples are ground to a fine powder, defatted twice with hexane, and extracted with suitable assay buffer (for example 5 M urea, 2 M thiourea, 50 mM Tris (pH 8.0), 65 mM DTT). The samples are sonicated in buffer to extract proteins. The extracted proteins are diluted, denatured, and then proteolytically digested by adding trypsin protease and incubating at 37°C for 15-20 hours. The digestion reactions are acidified with formic acid (pH = 1 -2) and are analyzed using LC-MS/MS.

The selected signature peptides can be used for both qualitative and quantitative analysis of Gly m 4, either by itself or in combination with additional proteins in a multiplexing assay format. In this example, several signature peptides are selected from all peptide possibilities (SEQ ID NO: 52 MGVFTFEDEINSPVAPATLYK; SEQ ID NO: 53 ALDSFK; SEQ ID NO: 54 SVENVEGNGGPGTIK; SEQ ID NO: 55 ITFLEDGETK; SEQ ID NO: 56 FVLHK; and SEQ ID NO: 57 AIEAYLLAHPDYN), and representative quantitation of these signature peptides are shown in FIGs. 4A - 4F. Synthetic peptides can directly serve as an analytical reference standard for protein quantitation. Example 5

Several homologous protein sequences for Gly m 5 are identified from public databases including NCBI, Phytozome, and UniProt. Identified sequences (SEQ ID NOs: 1 5 and 62-74) are analyzed using bioinformatics tools to identify sequence homology and shared sequence composition among the available protein sequences (see FIG. 5W). Specifically this involved the use of Vector NTI Align X alignment tool which performs a CLUSTAL W type alignment. From this analysis, a consensus sequence and/or representative sequence can be determined.

Once the consensus sequence and/or representative sequence is chosen or determined, it is digested in silico to generate candidate signature peptide fragments to be detected and measured by LC-MS. According to the unique approaches provided herein, signature peptides are selected based on the degree of conservation among the available protein sequences, such that the selected signature peptide can be used to quantify al l or as many protein isoforms as possible among the identified protein sequences found in the public sequence databases. As a result, quantitation of selected signature peptides can not only measure Gly m 5 itself, but also measure potential allergens which are highly homologous to Gly m 5.

The selected signature peptides can be used for both qualitative and quantitative analysis of Gly m 5, either by itself or in combination with additional proteins in a multiplexing assay format. In this example, several signature peptides are selected from all peptide possibilities (SEQ ID NO: 3 NILEASYDTK; SEQ ID NO: 75 CNLLK; SEQ ID NO: 76 EEDEDEQPRPIPFPRPQPR; SEQ ID NO: 77 EEQEWPR; SEQ ID NO: 78 QFPFPRPPHQK; SEQ ID NO: 79 ESEESEDSELR; SEQ ID NO: 80 NPFLFGSNR; SEQ ID NO: 81 FETLFK; SEQ ID NO: 82 SPQLQNLR; SEQ ID NO: 83 LQSGDALR; SEQ ID NO: 84 VPSGTTYYVVNPDNNENLR; SEQ ID NO: 85 FESFFLSSTEAQQSYLQGFSR; SEQ ID NO: 86 FEEINK; SEQ ID NO: 87 VLFSR; SEQ ID NO: 88 TISSEDKPFNLR; SEQ ID NO: 89 DP1YSNK; SEQ ID NO: 90 FFEITPEK; SEQ ID NO: 91 AIVILVINEGDANIELVGLK; SEQ ID NO: 92 EQQQEQQQEEQPLEVR; SEQ ID NO: 93

NFLAGSQDNVISQIPSQVQELAFPGSAQAVEK; and SEQ ID NO: 94 ESYFVDAQPK), and representative quantitation of these signature peptides are shown in FIGs. 5A - 5U. A peptide standard is synthesized for SEQ ID NO: 3 NILEASYDTK for quantitative and qualitative analyses (see FIG. 5V). Synthetic peptides can directly serve as an analytical reference standard for protein quantitation.

Example 6

At least two homologous protein sequences for Gly m 6 Gl are identified from public databases including NCBI, Phytozome, and UniProt. Identified sequences (SEQ ID NOs: 16 and 95) are analyzed using bioinformatics tools to identify sequence homology and shared sequence composition among the available protein sequences (see FIG. 6L). Specifically this involved the use of Vector NTI Align X alignment tool which performs a CLUSTAL W type alignment. From this analysis, a consensus sequence and/or representative sequence can be determined.

Once the consensus sequence and/or representative sequence is chosen or determined, it is digested in silico to generate candidate signature peptide fragments to be detected and measured by LC-MS. According to the unique approaches provided herein, signature peptides are selected based on the degree of conservation among the available protein sequences, such that the selected signature peptide can be used to quantify all or as many protein isoforms as possible among the identified protein sequences found in the public sequence databases. As a result, quantitation of selected signature peptides can not only measure Gly m 6 Gl itself, but also measure potential allergens which are highly homologous to Gly m 6 Gl .

The selected signature peptides can be used for both qualitative and quantitative analysis of Gly m 6 Gl , either by itself or in combination with additional proteins in a multiplexing assay format. In this example, several signature peptides are selected from all peptide possibilities (SEQ ID NO: 96 LVFSLCFLLFSGCCFAFSSR; SEQ ID NO: 97 EQPQQNECQIQK; SEQ ID NO: 98 RPSYTNGPQEIYIQQGK; SEQ ID NO: 99 HQQEEENEGGSILSGFTLEFLEHAFSVDK; SEQ ID NO: 100 HCQRPR; SEQ ID NO: 101 HNIGQTSSPDIYNPQAGSVTTATSLDFPALSWLR; SEQ ID NO: 102 ALIQVVNCNGER; SEQ ID NO: 103 VFDGELQEGR; SEQ ID NO: 104 TNDTPMIGTLAGANSLLNALPEEVIQHTFNLK; SEQ ID NO: 105 NN PFK; and SEQ ID NO: 106 FLVPPQESQK), and representative quantitation of these signature peptides are shown in FIGs. 6A - 6K. Synthetic peptides can directly serve as an analytical reference standard for protein quantitation.

Example 7

At least two homologous protein sequences for Gly m 6 G2 are identified from public databases including NCBL Phytozome, and UniProt. Identified sequences (SEQ ID NOs: 17 and 107) are analyzed using bioinformatics tools to identify sequence homology and shared sequence composition among the available protein sequences (see FIG. 7P). Specifically this involved the use of Vector NTI Align X alignment tool which performs a CLUSTAL W type alignment. From this analysis, a consensus sequence and/or representative sequence can be determined.

Once the consensus sequence and/or representative sequence is chosen or determined, it is digested in silico to generate candidate signature peptide fragments to be detected and measured by LC-MS. According to the unique approaches provided herein, signature peptides are selected based on the degree of conservation among the available protein sequences, such that the selected signature peptide can be used to quantify all or as many protein isoforms as possible among the identified protein sequences found in the public sequence databases. As a result, quantitation of selected signature peptides can not only measure Gly m 6 G2 itself, but also measure potential allergens which are highly homologous to Gly m 6 G2.

The selected signature peptides can be used for both qualitative and quantitative analysis of Gly m 6 G2, either by itself or in combination with additional proteins in a multiplexing assay format. In this example, several signature peptides are selected from all peptide possibilities (SEQ ID NO: 4 VTAPAMR; SEQ ID NO: 108 LVLSLCFLLFSGCFALR; SEQ ID NO: 109 EQAQQNECQIQK; SEQ ID NO: 1 10 RPSYTNGPQE1Y1QQGNGIFGMIFPGCPSTYQEPQESQQR; SEQ ID NO: 1 1 1 SQRPQDR; SEQ ID NO: 1 12 QQEEENEGSNILSGFAPEFLK; SEQ ID NO: 1 13 EAFGVNMQIVR; SEQ ID NO: 1 14 PQQEEDDDDEEEQPQCVETDK; SEQ ID NO: 1 15 LSAQYGSLR; SEQ ID NO: 1 16 NAMFVPHYTLNANSIIYALNGR; SEQ ID NO: 1 17 ALVQVVNCNGER; SEQ ID NO: 1 18 VFDGELQEGGVLIVPQNFAVAA ; SEQ ID NO: 1 19 TNDRPSIGNLAGANSLLNALPEE-VIQHTFNLK; and SEQ ID NO: 120 NNNPFSFLVPPQESQR), and representative quantitation of these signature peptides are shown in FIGs. 7A - 7N. A peptide standard is synthesized for SEQ ID NO: 4 VTAPAMR for quantitative and qualitative analyses (see FIG. 70). Synthetic peptides can directly serve as an analytical reference standard for protein quantitation.

Example 8

The Gly m 6 G3 sequence identified from databases is digested in silico to generate candidate signature peptide fragments to be detected and measured by LC-MS. According to the unique approaches provided herein, signature peptides are selected based on the degree of conservation among the available protein sequences, such that the selected signature peptide can be used to quantify all or as many protein isoforms as possible among the identified protein sequences found in the public sequence databases. Quantitation of selected signature peptides can not only measure Gly m 6 G3 itself, but also measure potential allergens which are highly homologous to Gly m 6 G3.

The selected signature peptides can be used for both qualitative and quantitative analysis of Gly m 6 G3, either by itself or in combination with additional proteins in a multiplexing assay format. In this example, several signature peptides are selected from all peptide possibilities (SEQ ID NO: 5 NNNPFSFLVPPK; SEQ ID NO: 121 LVLSLCFLLFSGCCFAFSFR; SEQ ID NO: 122 EQPQQNECQIQR; SEQ ID NO: 123 QQEEENEGGSILSGFAPEFLEHAFVVDR; SEQ ID NO: 124 LQGENEEEEK; SEQ ID NO: 125 GGLSVISPPTEEQQQRPEEEEKPDCDEK; SEQ ID NO: 126 HCQSQSR; SEQ ID NO: 127 LSAQFGSLR; SEQ ID NO: 128 VFDGELQEGQVLIVPQNFAVAAR; and SEQ ID NO: 129 TNDRPSIGNLAGANSLLNALPEEVIQQTFNLR), and representative quantitation of these signature peptides are shown in FIGs. 8A - 8J. A peptide standard is synthesized for SEQ ID NO: 5 NNNPFSFLVPPK for quantitative and qualitative analyses (see FIG. 8K). Synthetic peptides can directly serve as an analytical reference standard for protein quantitation. Example 9

Several homologous protein sequences for Gly m 6 G4 are identified from public databases including NCBI, Phytozome, and UniProt. Identified sequences (SEQ ID NOs: 19 and 131 -134) are analyzed using bioinformatics tools to identify sequence homology and shared sequence composition among the available protein sequences (see FIG. 9Y). Specifically this involved the use of Vector NTI Align X alignment tool which performs a CLUSTAL W type alignment. From this analysis, a consensus sequence and/or representative sequence can be determined.

Once the consensus sequence and/or representative sequence is chosen or determined, it is digested in silico to generate candidate signature peptide fragments to be detected and measured by LC-MS. According to the unique approaches provided herein, signature peptides are selected based on the degree of conservation among the available protein sequences, such that the selected signature peptide can be used to quantify all or as many protein isoforms as possible among the identified protein sequences found in the public sequence databases. As a result, quantitation of selected signature peptides can not only measure Gly m 6 G4 itself, but also measure potential allergens which are highly homologous to Gly m 6 G4.

The selected signature peptides can be used for both qualitative and quantitative analysis of Gly m 6 G4, either by itself or in combination with additional proteins in a multiplexing assay format. In this example, several signature peptides are selected from all peptide possibilities (SEQ ID NO: 6 ADFYNPK; SEQ ID NO: 135 LNECQLNNLNALEPDHR; SEQ ID NO: 136 CAGVTVSK; SEQ ID NO: 137 LTLNR; SEQ ID NO: 138 MIIIAQGK; SEQ ID NO: 139 GALGVAIPGCPETFEEPQEQSNR; SEQ ID NO: 140 QQLQDSHQK; SEQ ID NO: 141 VFYLAGNPDIEYPETMQQQQQQK; SEQ ID NO: 142 QGQHQQEEEEEGGSVLSGFSK; SEQ I D NO: 143 HFLAQSFNTNEDIAEK; SEQ ID NO: 144 Q1VTVEGGLSV1SPK; SEQ ID NO: 145 WQEQQDEDEDEDEDDEDEQ1 PSHPPR; SEQ I D NO: 146 RPSHG ; SEQ ID NO: 147 EQDEDEDEDEDKPRPSRPSQGK; SEQ ID NO: 148 QEEPR; SEQ ID NO: 149 NGVEENICTLK; SEQ ID NO: 150 LHENIARPSR; SEQ ID NO: 151 ISTLNSLTLPALR; SEQ ID NO: 152 QFQLSAQYVVLYK; SEQ ID NO: 153 NGIYSPHWNLNANSV1YVTR; SEQ ID NO: 154 DVFR; SEQ ID NO: 155 AIPSEVLAHSY LR; SEQ ID NO: 156 QSQVSELK; and SEQ ID NO: 58 YEGNWGPLVNPESQQGSPR), and representative quantitation of these signature peptides are shown in FIGs. 9A - 9X. Synthetic peptides can directly serve as an analytical reference standard for protein quantitation. Example 10

The Gly m 6 precursor is digested in silico to generate candidate signature peptide fragments to be detected and measured by LC-MS. According to the unique approaches provided herein, signature peptides are selected based on the degree of conservation among the available protein sequences, such that the selected signature peptide can be used to quantify all or as many protein isoforms as possible among the identified protein sequences found in the public sequence databases. As a result, quantitation of selected signature peptides can not only measure Gly m 6 precursor itself, but also measure potential allergens which are highly homologous to Gly m 6 precursor.

The selected signature peptides can be used for both qualitative and quantitative analysis of Gly m 6 precursor, either by itself or in combination with additional proteins in a multiplexing assay format. In this example, three signature peptides are selected from all peptide possibilities (SEQ ID NO: 6 ADFYNPK, SEQ ID NO: 59 GALQCKPGCPETFEEPQEQSNR and SEQ ID NO: 60 LQSPDDER), and representative quantitation of these signature peptides are shown in FIGs. 10B and I OC. Synthetic peptides can directly serve as an analytical reference standard for protein quantitation. Sequence Listing

SEQ ID NO: 1 Exemplary signature peptide for Gly m 1

SYPSNATCPR

SEQ ID NO: 2 Exemplary signature peptide for Gly m 3

YMVIQGEPGAVIR

SEQ ID NO: 3 Exemplary signature peptide for Gly m 5 (beta-conglycinin) NILEASYDTK

SEQ ID NO: 4 Exemplary signature peptide for Gly m 6 (Glycinin) G2

VTAPAMR SEQ ID NO: 5 Exemplary signature peptide for Gly m 6 (Glycinin) G3

NNNPFSFLVPPK

Exemplary signature peptide for Gly m 6 (Glycinin) precur; Exemplary signature peptide for Kunitz trypsin inhibitor 1

SEQ ID NO: 8 Exemplary signature peptide for Kunitz trypsin inhibitor 3

GIGTIISSPYR

SEQ ID NO: 9 Exemplary signature peptide for Gly m Bd 28 K

NKPQFLAGAASLLR SEQ ID NO: 10 Exemplary signature peptide for Gly m Bd 30 K

GVITQVK

SEQ ID NO: 1 1 Exemplary signature peptide for Gly m 8 (2S albumin)

IMENQSEELEEK

SEQ ID NO: 12 Gly m 1 ABA54898.1 [MW= 1 2482.64 Da]

MGSKVVASVALLLSINILFISMVSSSSHYDPQPQPSHVTALITRPSCPDLSICLNILGG SLGTVDDCCALIGGLGDIEAIVCLCIQLRALGILNLNRNLQLILNSCGRSYPSNATCP RT

SEQ ID NO: 13 Gly m 3 CAA 1 1 755.1 [MW= 1 41 00.07 Da]

MSWQAYVDDHLLCGIEGNHLTHAAIIGQDGSVWLQSTDFPQFKPEEITAIMNDFNE

PGSLAPTGLYLGGTKYMV1QGEPGAVIRGKKGPGGVTVKKTGAALI1GIYDEPMTP

GQCN VVERLGDYLIDQGY

SEQ I D NO: 14 Gly m 4 P26987 [MW= 1 6771 .81 Da] MGVFTFEDEINSPVAPATLYKALVTDADNVIPKALDSFKSVENVEGNGGPGTIKKI TFLEDGETKFVLHKIESIDEANLGYSYSVVGGAALPDTAEK1TFDSKLVAGPNGGS AGKLTVKYETKGDAEPNQDELKTGKAKADALFKAIEAYLLAHPDY SEQ ID NO: 15 Gly m 5 (beta-conglycinin) 121281 [MW= 70293.13 Da]

MMRARFPLLLLGLVFLASVSVSFGIAYWEKENPKHNKCLQSCNSERDSYRNQACH ARCNLLKVEKEECEEGEIPRPRPRPQHPEREPQQPGEKEEDEDEQPRPIPFPRPQPRQ EEEHEQREEQEWPRKEEKRGEKGSEEEDEDEDEEQDERQFPFPRPPHQKEERNEEE DEDEEQQRESEESEDSELRRHKNKNPFLFGSNRFETLFKNQYGRIRVLQRFNQRSP QLQNLRDYRILEFNSKPNTLLLPNHADADYLIVILNGTAILSLVNNDDRDSYRLQSG DALRVPSGTTYYVVNPDN ENLRLITLAIPVNKPGRFESFFLSSTEAQQSYLQGFSR NILEASYDTKFEEINKVLFSREEGQQQGEQRLQESVIVEISKEQIRALSKRAKSSSRK TISSEDKPFNLRSRDPIYSNKLGKFFEITPEKNPQLRDLDIFLSIVDMNEGALLLPHFN SKAIVILVINEGDANIELVGLKEQQQEQQQEEQPLEVRKYRAELSEQDIFVIPAGYP VVVNATSNLNFFAIG1NAEN QRNFLAGSQDNVISQIPSQVQELAFPGSAQAVEKL LKNQRESYFVDAQPKKKEEGNKGRKGPLSSILRAFY

SEQ ID NO: 16 Gly m 6 Glycinin Gl 121276 [MW= 55706.34 Da]

MAKLVFSLCFLLFSGCCFAFSSREQPQQNECQIQKLNALKPDNRIESEGGLIETWNP NNKPFQCAGVALSRCTLNRNALRRPSYTNGPQEIYIQQGKGIFGMIYPGCPSTFEEP QQPQQRGQSSRPQDRHQKIYNFREGDLIAVPTGVAWWMYNNEDTPVVAVSIIDTN SLENQLDQMPRRFYLAGNQEQEFLKYQQEQGGHQSQKGKHQQEEENEGGSILSGF TLEFLEHAFSVDKQIAKNLQGENEGEDKGAIVTVKGGLSVIKPPTDEQQQRPQEEE EEEEDEKPQCKGKDKHCQRPRGSQSKSRRNGIDETICTMRLRHNIGQTSSPDIYNPQ AGSVTTATSLDFPALSWLRLSAEFGSLRKNAMFVPHYNLNANSIIYALNGRALIQV VNCNGERVFDGELQEGRVLIVPQNFVVAARSQSDNFEYVSFKTNDTPMIGTLAGA NSLLNALPEEVIQHTFNLKSQQARQIKNNNPFKFLVPPQESQKRAVA

SEQ ID NO: 17 Gly m 6 Glycinin G2 121277 [MW= 54390.76 Da]

MAKLVLSLCFLLFSGCFALREQAQQNECQIQKLNALKPDNRIESEGGFIETWNPNN KPFQCAGVALSRCTLNRNALRRPSYTNGPQEIYIQQGNGIFGMIFPGCPSTYQEPQE SQQRGRSQRPQDRHQKVHRFREGDLIAVPTGVAWWMYNNEDTPVVAVSIIDTNS LENQLDQMPRRFYLAGNQEQEFLKYQQQQQGGSQSQKGKQQEEENEGSN1LSGF APEFLKEAFGVNMQIVRNLQGENEEEDSGAIVTVKGGLRVTAPAMRKPQQEEDDD DEEEQPQCVETDKGCQRQSKRSRNGIDET1CTMRLRQNIGQNSSPD1YNPQAGSITT ATSLDFPALWLLKLSAQYGSLRKNAMFVPHYTLNANSIIYALNGRALVQVVNCNG ERVFDGELQEGGVLIVPQNFAVAAKSQSDNFEYVSFKTNDRPS1GNLAGANSLLNA LPEEVIQHTFNLKSQQARQVKNNNPFSFLVPPQESQRRAVA SEQ ID NO: 18 Gly m 6 Glycinin G3 121278 [MW= 54241.73 Da]

MAKLVLSLCFLLFSGCCFAFSFREQPQQNECQIQRLNALKPDNRIESEGGFIETWNP NNKPFQCAGVALSRCTLNRNALRRPSYTNAPQE1YIQQGSGIFGM1FPGCPSTFEEP QQKGQSSRPQDRHQKIYHFREGDLIAVPTGFAYWMYNNEDTPVVAVSLIDTNSFQ NQLDQMPRRFYLAGNQEQEFLQYQPQKQQGGTQSQ GKRQQEEENEGGSILSGF APEFLEHAFVVDRQ1VRKLQGENEEEEKGA1VTVKGGLSVISPPTEEQQQRPEEEEK PDCDEKDKHCQSQSRNG1DETICTMRLRHNIGQTSSPD1FNPQAGSITTATSLDFPAL SWL LSAQFGSLRKNAMFVPHYNLNANSIIYALNGRALVQVVNCNGERVFDGEL QEGQVLIVPQNFAVAARSQSDNFEYVSFKTNDRPSIGNLAGANSLLNALPEEV1QQ TFNLRRQQARQV NNNPFSFLVPPKESQRRVVA SEQ ID NO: 19 Gly m 6 Glycinin G4 121279 [MW= 63587.16 Da]

MGKPFTL SLS SLCLLLLS S ACF AI S S SKLNEC QLN LN ALEPDHRVE SEGGLIQTWN SQHPELKCAGVTVSKLTLNRNGLHSPSYSPYPRMIIIAQGKGALGVAIPGCPETFEE PQEQSNRRGSRSQKQQLQDSHQKIRHFNEGDVLVIPPSVPYWTY TGDEPVVAISL LDTSNFNNQLDQTPRVFYLAGNPDIEYPETMQQQQQQKSHGGRKQGQHQQEEEE EGGSVLSGF SKHFLAQSFNTNEDI AEKLE SPDDERKQI VTVEGGLS VI SPK WQEQQ DEDEDEDEDDEDEQIPSHPPRRPSHGKREQDEDEDEDEDKPRPSRPSQGKRNKTGQ DEDEDEDEDQPRKSREWRSKKTQPRRPRQEEPRERGCETRNGVEENICTLKLHENI ARPSRADFYNPKAGRISTLNSLTLPALRQFQLSAQYVVLYK GIYSPHWNLNANSV

lYVTRGQGKVRVVNCQGNAVFDGELRRGQLLVVPQNFVVAEQAGEQGFEYIVFK THHNAVTSYLKDVFRAIPSEVLAHSY LRQSQVSELKYEGNWGPLV PESQQGSP RVKVA SEQ ID NO: 20 Gly m 6 Glycinin precursor 75221455 [MW= 63876.47 Da]

MGKPFTLSLS SLCLLLLS S ACF AI S SSKLNECQLNNLNALEPDHRVEFEGGLIQTWN SQHPELKCAGVTVSKLTLNRNGLHLPSYSPYPRMIIIAQGKGALQCKPGCPETFEEP QEQ SNRRG SRS QKQQLQDSHQKIRHFNEGDVL VIPPG VP Y WTYNTGDEP V V AI SLL DTSNFNNQLDQTPRVFYLAGNPDIEYPETMQQQQQQKSHGGRKQGQHQQEEEEE GGSVLSGFSKHFLAQSFNTNEDIAEKLQSPDDERKQIVTVEGGLSVISPKWQEQQD EDEDEDEDDEDEQIPSHPPRRPSHGKREQDEDEDEDEDKPRPSRPSQGKREQDQDQ DEDEDEDEDQPRKSREWRSKKTQPRRPRQEEPRERGCETRNGVEENICTLKLHENI ARPSRADFYNPKAGRISTLNSLTLPALRQFQLSAQYVVLYKNGIYSPHWNLNANSV IYVTRGQGKVRVVNCQGNAVFDGELRRGQLLVVPQNFVVAEQAGEQGFEYIVFK THHNAVTSYLKDVFRAIPSEVLAHSYNLRQSQVSELKYEGNWGPLVNPESQQGSP RVKVA

SEQ ID NO: 21 Gly m Bd 28K 12697782 [MW= 52944.36 Da]

MGNKTTLLLLLFVLCHGVATTTMAFHDDEGGDKKSPKSLFLMSNSTRVFKTDAG EMRVLKSHGGRIFYRHMHIGFISMEPKSLFVPQYLDSNLIIF1RRGEAKLGFIYDDEL AERRLKTGDLYMIPSGSAFYLVNIGEGQRLHVICSIDPSTSLGLETFQSFYIGGGANS HSVLSGFEPAILETAFNESRTVVEEIFSKELDGPIMFVDDSHAPSLWTKFLQLKKDD KEQQLKKMMQDQEEDEEEKQTSRSWRKLLETVFGKVNEKIENKDTAGSPASYNL YDDKKADFKNAYGWSKALHGGEYPPLSEPDIGVLLVKLSAGSMLAPHVNPISDEY TIVLSGYGELHIGYPNGSRAMKTKIKQGDVFVVPRYFPFCQVASRDGPLEFFGFSTS ARKNKPQFLAGAASLLRTLMGPELSAAFGVSEDTLRRAVDAQHEAVILPSAWAAP PENAGKLKMEEEPNAIRSFANDVVMDVF

SEQ ID NO: 22 Gly m Bd 30K 84371705 [MW= 42757.81 Da]

MGFLVLLLFSLLGLSSSSSISTHRS1LDLDLTKFTTQKQVSSLFQLWKSEHGRVYHN HEEEAKRLEIFKNNLNYIRDMNANRKSPHSHRLGLNKFAD1TPQEFSKKYLQAPKD VSQQIKMANKKMKKEQYSCDHPPASWDWRKKGVITQVKYQGGCGSGWAFSATG AIEAAHAIATGDLVSLSEQELVDCVEESEGCYNGWHYQSFEWVLEHGGIATDDDY PYRAKEGRCKANKIQDKVTIDGYETLIMSDESTESETEQAFLSAILEQP1SVSIDAKD FHLYTGGIYDGENCTSPYGINHFVLLVGYGSADGVDYWIAKNSWGEDWGEDGYI WIQRNTGNLLGVCGMNYFASYPTKEESETLVSARVKGHRRVDHSPL

SEQ ID NO: 23 KTI 1 125722 [MW= 22545.94 Da] MKST1FFALFLVCAFTISYLPSATAQFVLDTDDDPLQNGGTYYMLPVMRGKGGGIE VDSTGKEICPLTVVQSPNELDKGIGLVFTSPLHALFIAERYPLSIKFGSFAVITLCAG MPTEWAIVEREGLQAVKLAARDTVDGWFNIERVSREYNDYKLVFCPQQAEDNKC EDIGIQIDDDGIRRLVLSKNKPLVVQFQKFRSSTA

SEQ ID NO: 24 KTI 3 125020 [ MW= 24005.29 Da]

MKSTIFFLFLFCAFTTSYLPSAIADFVLDNEGNPLENGGTYYILSDITAFGGIRAAPT GNERCPLTVVQSRNELDKGIGTIISSPYRIRFIAEGHPLSLKFDSFAVIMLCVGIPTEW SVVEDLPEGPAVKIGENKDAMDGWFRLERVSDDEFNNYKLVFCPQQAEDDKCGD IGISIDHDDGTRRLVVSKNKPLVVQFQKLDKESLAKKNHGLSRSE

SEQ ID NO: 25 Gly m 8 (2S albumin) NP_001238443 [MW= 18459.97 Da] MTKFTILLISLLFCIAHTCSASKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGD DDDDDDDDNHILRTMRGRINYIRRNEGKDEDEEEEGHMQKCCTEMSELRSPKCQC KALQKIMENQSEELEEKQKKKMEKELINLATMCRFGPMIQCDLS SDD

SEQ ID NO : 26 Lectin ADC94422 [M W= 30186.22 Da]

MATSNFSIVLSLSLAFFLVLLTKANSTNTVSFTVSKFSPRQQNLIFQGDAAISPSGVL RLTKVDSIDVPTTGSLGRALYATPIQIWDSETGKVASWATSFKFKVFSPNKTADGL AFFLAPVGSKPQSKGGFLGLFNSDSKNKSVQTVAVEFDTYY AKWDPANRHIGID VNSIKSVKTASWGLANGQIAQILITYDADTSLLVASLIHPSRKTSYILSETVSLKSNL PEWVNIGFSATTGLNKGFVETHDVFSWSFASKLSDGSTSDTLDLPSFLLNEAI

SEQ ID NO: 27 Lipoxygenase CAA39604 [MW= 96817.14 Da]

MFGIFDKGQKIKGTVVLMPKNVLDFNAITSIGKGGVIDTATGILGQGVSLVGGVID TATSFLGRNISMQLISATQTDGSGNGKVGKEVYLEKHLPTLPTLGARQDAFSIFFE WDASFGIPGAFYIKNFMTDEFFLVSVKLEDIPNHGTIEFVCNSWVYNFRSYKKNRIF FVNDTYLPSATPAPLLKYRKEELEVLRGDGTGKRKDFDRIYDYDVYNDLGNPDGG DPRPILGGSSIYPYPRRVRTGRERTRTDPNSEKPGEVYVPRDENFGHLKSSDFLTYG IKSLSHDVIPLFKSAIFQLRVTSSEFESFEDVRSLYEGGIKLPTDILSQISPLPALKEIFR TDGENVLQFPPPHVAKVSKSGWMTDEEFAREVIAGVNPNVIRRLQEFPPKSTLDPT LYGDQTSTITKEQLEINMGGVTVEEALSTQRLFILDYQDAFIPYLTRINSLPTAKAY ATRTILFLKDDGTLKPLAIELSKPHPDGDNLGPESIVVLPATEGVDSTIWLLAKAHV IVNDSGYHQLVSHWLNTHAVMEPFAIAT RHLSVLHPIYKLLYPHYRDTININGLA RQSLINADGIIEKSFLPGKYSIEMSSSVYKNWVFTDQALPADLVKRGLAIEDPSAPH GLRLVIEDYPYAVDGLEIWDAIKTWVHEYVSLYYPTDAAVQQDTELQAWWKEA VEKGHGDLKEKPWWPKMQTTEDLIQSCSIIVWTASALHAAVNFGQYPYGGLILNR PTLARRFIPAEGTPEYDEMVKNPQKAYLRTITPKFETLIDLSVIEILSRHASDEIYLGE RETPNWTTD KALEAF RFGSKLTGIEGK1NARNSDPSLRNRTGPVQLPYTLLHRS SEEG LTFKGIPN SI SI

SEQ ID NO: 28 Gly m 1 consensus sequence

MGSKVVASVALLLS1N1LFISMVSSSSHYDPPQPSYVTALITRPSCPDLSICLNILGGS LGTVDDCCALIGGLGDIEAIVCLCIQLRALGILNLNRNLQLILNACGRSYPSNATCP RT

SEQ ID NO: 29 Gly m 1 consensus sequence #2 MGSKVVASVALLLSINILFISMVSSSSHYDPQPQPSHVTALITRPSCPDLSICLNILGG SLGTVDDCCALIGGLGDIEAlVCLCIQLPvALGILNLNRNLQLILNACGRSYPSNATCP RT SEQ ID NO: 30 Gly m 1 Ping AAB34755.1 42aa

ALITRPSXPDLSIXLNILGGSLGTVDDXXALIGGLXDXXAIV

SEQ ID NO: 31 Gly m 1 Ping ABA54897.1 134aa

MGSKVVASVALLLSINILFISMVSSSSHYDPPPPPCYVPAPLTPPPSLSPPPSLSPPPPS GPSCPDLSVCLNILDGSPADDCC ALIADLVDLEASVCLCIQLRVLGIVNLDLNLQLI LNACGPSYPSNATCPRT

SEQ ID NO: 32 Gly m 1 Eric ABA54899 1 18aa

GSKVVASVALLLSINILFISMVSSSSHYDPQPQPSHVTALITRPSCPDLSICLNILGGS LGTVDDCCALIGGLGDIEAIVCLCIQLRALGILNLNRNLQLILNSCGRSYPSNATCPR T

SEQ ID NO: 33 Gly m 1 Glymal 5gl3740.1 BLAST 120aa

MGSKVVAYVALLLSINILFISMVSSSSHYDPQPQPSYVTALITRPSCPDLSVCLNILG GYLGTVDDCCALIGGLGDIEATVCLCIQLRALGILNLNRNLQLILNACGPSYPSNAT CPRT

SEQ ID NO: 34 Gly m 1 Glymal 5gl 3770.1 BLAST 130aa

MGSKVVASVALLLSINILFISMVSSSSHYDPPPPPCYVPAPFTPPPPSLSPPPPSGPSCP DLSVCLNILDGSPADDCCALIADLVDLEASVCLCIQLRVLGIVNLDLNLQLILNACG PSYPSNATCPRT

SEQ ID NO: 35 Gly m 1 Glymal 5gl3750.1 BLAST 120aa

MGSKVVASVALLLSINILFISMVSSSSHYDPPPQPSYVTALITRPSCPDLSICLNILGG SLGTVDDCCALIGGLGDIEAIVCLCIQLRALGILNLNRNLQLILNSCGRSYPSNATCP RT

SEQ ID NO: 36 ALGILNLNR

SEQ ID NO: 37 NLQLILNSCGR

SEQ ID NO: 38 Gly m 3 consensus sequence

MSWQAYVDDHLLCGIEGNHLTHAAIIGQDGSVWAQSTDFPQFKPEEITAIMNDFN EPGSLAPTGLYLGGTKYMVIQGEPGAVIRGKKGPGGVTVKKTGAALIIGIYDEPMT PGQCNMVVERLGDYLIDQGY

SEQ ID NO: 39 Gly m 3 Ping ABU97472.1 131aa

MSWQAYVDDHLLCEIEGNHLTHAAIIGQDGSVWAQSTNFPQFKPEEITAINNDFNE PGSLAPTGLYIGGTKYMVIQGEPGAVIRGKKGPGGVTVKKTGAALIIGIYDEPMTP GQCNMVVERLGDYLIDQGL

SEQ ID NO: 40 Gly m 3 Ping 065809.1 131 aa MSWQAYVDDHLLCDIEGNHLTHAAIIGQDGSVWAQSTDFPQFKPEEITAIMNDFN

EPGSLAPTGLYLGGTKYMVIQGEPGAV1RGKKGPGGVTVKKTGAALIIGIYDEPMT

PGQCNMVVERPGDYLIDQGY SEQ ID NO: 41 GPGGVTVK

SEQ ID NO: 42 Gly m 4 consensus sequence

MGVFTFEDETTSPVAPATLYKALVTDADNVIPKAVDAFKSVENVEGNGGPGTIKKI TFVEDGETKFVLHKIEA1DEANLGYSYSVVGGAGLPDTVEKITFEAKLAAGANGGS AGKLTVKYQTKGDAQPNQDELKSGKAKADALFKAVEAYLLANPDYN

SEQ ID NO: 43 Gly m 4 Glyma07g37240.1 BLAST 165aa

MGVFTFEDEINSPVAPATLYKALVTDADNVIPKALDSFKSVENVEGNGGPGTIKKI TFLEADVNEWIDGETKFVLHKIESIDEANLGYSYSVVGGAALPDTAEKITFDSKLV AGPNGGSAGKLTVKYETKGDAEPNQDELKTGKAKADALFKAIEAYLLAHPDYN

SEQ ID NO: 44 Gly m 4 Glymal 7g03365.1 BLAST 159aa

MGIFTFEDEITSPVAPATLYKALVTDADNIIPKALDSFKSVENVEGNGGPGTIKKITF VEDGETKFVLHKIEAVDEANLGYSYSVVGGAALPDTAEKITFHSKLAAGPNGGSA GKLTVEYQTKGDAQPNQDQLKTGKAKADALFKAIEAYLLANPDYN

SEQ ID NO: 45 Gly m 4 Glyma07g37240.3 BLAST 147aa

MGVFTFEDEINSPVAPATLYKALDSFKSVENVEGNGGPGTIKKITFLEDGETKFVLH KIESIDEANLGYSYSVVGGAALPDTAEKITFDSKLVAGPNGGSAGKLTVKYETKGD AEPNQDELKTGKAKADALFKAIEAYLLAHPDYN

SEQ ID NO: 46 Gly m 4 Glyma07g37270.2 BLAST 159aa

MGVFTFEDEINSPVAPATLYKALVTDADNVIPKALDSFKSVENVEGNGGPGTIKKI TFLEDGETKFVLHKIEAIDEANLGYSYSVVGGDGLPDTVEKITFECKLAAGANGGS AGKLTVKYQTKGDAQPNQDDLK1GKAKSDALFKAVEAYLLAHPDYN

SEQ ID NO: 47 Gly m 4 Glyma07g37270.1 BLAST 159aa

MGVFTFEDETTSPVAPATLYKALVTDADNVIPKAVDAFRSVENVEGNGGPGTIKKI TFLEDGETKFVLHKIEAIDEANLGYSYSVVGGDGLPDTVEKITFECKLAAGANGGS AGKLTVKYQTKGDAQPNQDDLKIGKAKSDALFKAVEAYLLAHPDYN

SEQ ID NO: 48 Gly m 4 Glymal 7g03360.1 BLAST 159aa

MGVFTFEDETTSPVAPATLYKALVTDADNVIPKAVDAFRSVENLEGNGGPGTIKKI TFVEDGESKFVLHKIESVDEANLGYSYSVVGGVGLPDTVEKITFECKLAAGANGGS AGKLTVKYQTKGDAQPNPDDLKIGKVKSDALF AVEAYLLANPHYN

SEQ ID NO: 49 Gly m 4 Glymal 7g03350.1 BLAST 159aa

MGIFTFEDETTSPVAPATLYKALVTDADNVIPKAVEAFRSVENLEGNGGPGTIKKIT FVEDGES FVLHKIESVDEANLGYSYSVVGGVGLPDTVEKITFECKLAAGANGGS AGKLTVKYQTKGDAQPNPDDLK1GKVKSDALFKAVEAYLLANPHYN

SEQ ID NO: 50 Gly m 4 Glyma09g04510.1 BLAST 1 59aa MGVFTFEDETTSTVAPARLYKALVKDADNLVPKAVEAIKSVEIVEGNGGPGTIKKL

TFVEDGQTKYVLHKVEAIDEANWGYNYSVVGGVGLPDTVEKISFEAKLVADPNG

GSIAK1TVKYQTKGDANPSEEELKSGKAKGDALFKALEGYVLANPDYN

SEQ ID NO: 51 Gly ra 4 Glymal 5gl 5590.1 BLAST 159aa

MGVFTFEDETTSTVAPARLYKALVKDADNLVPKAVEAIKSVEIVEGNGGPGTIKKL

TFVEDGQTKYVLHKVEAIDEANWGYNYSVVGGVGLPDTVEKISFEAKLVEGASG

GSIAKITVKYQTKGDVNPSEEELKSGKAKGDALFKALEGYVLANPDYN

SEQ ID NO: 52 MGVFTFEDEINSPVAPATLYK

SEQ ID NO: 53 ALDSFK

SEQ ID NO: 54 SVENVEGNGGPGTIK

SEQ ID NO: 55 ITFLEDGETK

SEQ ID NO: 56 FVLHK

SEQ ID NO: 57 AIEAYLLAHPDYN

SEQ ID NO: 61 Gly m 5 consensus sequence

MMRARFPLLLLG V VFL A S V S V SFGI A Y WEKQNPSHNKCLQ SCN SEKD S YRNQ ACH ARCNLLKVEEEEECEEGQIPRPRPQHPERERQQHGEKEEDEGEQPRPFPFPRPRQPH QEEEHEQKEEHEWHRKEEKHGGKGSEEEQDEREHPRPHQPHQKEEEKHEWQHKQ EKHQGKESEEEEEDQDEDEEQDKESQESEGSESQREPRRHKNKNPFHFNSKRFQTL FKNQYGHVRVLQRFNKRSQQLQNLRDYRILEFNSKPNTLLLPHHADADYLIVILNG TAILTLVNNDDRDSYNLQSGDALRVPAGTTYYVVNPDNDENLRMITLAIPVNKPG RFESFFLSSTQAQQSYLQGFSKNILEASYDTKFEErNKVLFGREEGQQQGEERLQES VIVEISKKQIRELSKRAKSSSRKTISSEDKPFNLRSRDPIYSNKLGKLFEITPEKNPQL RDLDVFLSVVDMNEGALLLPHFNSKAIVVLVINEGDANIELVGIKEQQQRQQQEEQ PLEVRKYRAELSEQDIFVIPAGYPVVVNATSNLNFFAFGINAENNQRNFLAGSKDN VISQIPSQVQELAFPGSAKDIENLIKSQSESYFVDAQPQQKEEGNKGRKGPLSSILRA FY

SEQ ID NO: 62 Gly m 5 ABH09130 BLAST 600aa

MFGIVYWEKQNPSHNKCLRSCNSEKDSYRNQACHARCNLLKVEEEEECEEGQIPR

PRPQHPERERQQHGEKEEDEGEQPRPFPFPRPRQPHQEEEHEQKEEHEWHRKEEKH

GGKGSEEEQDEREHPRPHQPHQKEEEKHEWQHKQEKHQGKESEEEEEDQDEDEG QDKESQESEGSESQREPRRHKNKNPFHFNSKRFQTLFKNQYGHVRVLQRFNKRSQ QLQNLRDYRILEFNSKPNTLLSPNHADADYLIVILNGTAILTLVNNDDRDSYNLQS GDALRVPAGTTYYVVNPDNDENLRMITLAIPVNKPGRFESFFLSSTQAQQSYLQGF SKNILEASYDTKFEEINKVLFGREEGQQQGEERLQESVIVEISKKQIRELSKHAKPSS R TISSEDKPFNLRSRDPIYSNKLGKLFEITPEKNPQLRDLDVFLSVVDMNEGALFL PHFNSKAIVVLV1NEGEANIELVGIKEQQQRQQQEEQPLEVRKYRAELSEQDIFVIP AGYPVVVNATSDLNFFAFGINAENNQRNFLAGSKDNVISQIPSQVQELAFPGSAKD IENLIKSQSESYFVDAQPQQKEEGNKGRKGPLSSILRAFY

SEQ ID NO: 63 Gly m 5 BAA 74452 BLAST 559aa VEEEEECEEGQIPRPRPQHPERERQQHGEKEEDEGEQPRPFPFPRPRQPHQEEEHEQ KEEHEWHRKEEKHGGKGSEEEQDEREHPRPHQPHQKEEEKHEWQHKQEKHQGK ESEEEEEDQDEDEEQDKESQESEGSESQREPRRHKNKNPFHFNSKRFQTLFKNQYG HVRVLQRFNKRSQQLQNLRDYR1LEFNSKPNTLLLPHHADADYLIVILNGTAILTLV NNDDRDSY LQSGDALRVPAGTTYYVV PDNDENLRMITLAIPVNKPGRFESFFLS STQ AQQ S YLQGF SKNILE A S YDTKFEEI KVLFGREEGQQQGEERLQES VI VEI SKK QIRELSKHAKSSSRKTISSEDKPFNLRSRDPIYSNKLGKLFEITPEKNPQLRDLDVFLS VVDMNEGALFLPHFNSKAIVVLVINEGEANIELVGIKEQQQRQQQEEQPLEVRKYR AELSEQDIFVIPAGYPVVVNATSDLNFFAFGINAEN QRNFLAGSKDNVISQIPSQV QELAFPGSAKDIENLIKSQSESYFVDAQPQQKEEGNKGRKGPLSSILRAFY

SEQ ID NO: 64 Gly m 5 BAC78524 BLAST 621aa

MMRARFPLLLLGVVFLASVSVSFGIAYWEKQNPSHNKCLRSCNSEKDSYRNQACH ARCNLLKVEEEEECEEGQIPRPRPQFIPERERQQHGEKEEDEGEQPRPFPFPRPRQPR QEEEHEQKEEHEWHRKEEKHGGKGSEEEQDEREHPRPHQPHQKEEEKHEWQHKQ EKHQGKESEEEEEDQDEDEEQDKESQESEGSESQREPRRFIK KNPFFIFNSKRFQTL FKNQYGHVRVLQRFNKRSQQLQNLRDYRILEFNSKPNTLLLPHHADADYLIVILNG TAILTLVNNDDRDSYNLQSGDALRVPAGTTYYVVNPDNDENLRM1TLAIPVNKPG RFESFFLSSTQAQQSYLQGFSKNILEASYDTKFEEINKVLFGREEGQQQGEERLQES VIVEISKKQIRELSKHAKSSSRKTISSEDKPFNLRSRDPIYSNKLGKLFEITPEKNPQL RDLDVFLSVVDMNEGALFLPHFNSKAIVVLVINEGEANIELVGIKEQQQRQQQEEQ PLEVRKYRAELSEQDIFVIPAGYPVVVNATSDLNFFAFGINAENNQRNFLAGSKDN VISQIPSQVQELAFPGSAKDIENLIKSQSESYFVDAQPQQKEEGNKGRKGPLSSILRA FY

SEQ ID NO: 65 Gly m 5 Glyma20g28460.1 BLAST 440aa

MMRVRFPLLVLLGTVFLASVCVSLKVREDENNPFYLRSSNSFQTLFENQNGRIRLL QRFNKRSPQLENLRDYRIVQFQSKPNTILLPHHADADFLLFVLSGRAILTLVNNDDR DSYNLHPGDAQRIPAGTTYYLVNPHDHQNLKIIKLAIPVNKPGRYDDFFLSSTQAQ QSYLQGFSHNILETSFHSEFEEINRVLFGEEEEQRQQEGVIVELSKEQIRQLSRRAKS SSRKTISSEDEPFNLRSRNPIYSNNFGKFFEITPEKNPQLRDLDIFLSSVDINEGALLLP HFNSKAIVILVINEGDANIELVGIKEQQQKQKQEEEPLEVQRYRAELSEDDVFVIPA AYPFVVNATSNLNFLAFGINAENNQRNFLAGEKDNVVRQIERQVQELAFPGSAQD VERLLKKQRESYFVDAQPQQKEEGSKGRKGPFPSILGALY

SEQ ID NO: 66 Gly m 5 Ping AAB23463 439aa

MMRVRFPLLVLLGTVFLASVCVSLKVREDENNPFYFRSSNSFQTLFENQNVR1RLL QRFNKRSPQLENLRDYRIVQFQSKPNTILLPHHADADFLLFVLSGRAILTLVNNDDR DSYNLHPGDAQRIPAGTTYYLVNPHDHQNLKIIKLAIPVNKPGRYDDFFLSSTQAQ QSYLQGFSHNILETSFHSEFEEINRVLFGEEEEQRQQEGVIVELSKEQIRQLSRRAKS SSRKTISSEDEPFNLRSRNPtYSNNFGKFFEITPEKNPQLRDLDIFLSSVDINEGALLLP HFNSKAIVILVINEGDANIELVGIKEQQQKQKQEEEPLEVQRYRAELSEDDVFVIPA AYPFVVNATSNLNFLAFGINAENNQRNFLAGEKDNVVRQIERQVQELAFPGSAQD VERLLKKQRESYFVDAQPQQKEEGSKGRKGPFPS1LGALY

SEQ ID NO: 67 Gly m 5 Glyma20g28650.1 BLAST 606aa

MMRARFPLLLLGLVFLASVSVSFGIAYWEKENP HNKCLQSCNSERDSYRNQACH ARCNLLKVEKEECEEGEIPRPRPRPQHPEREPQQPGEKEEDEDEQPRP1PFPRPQPRQ EEEHEQREEQEWPRKEEKRGEKGSEEEDEDEDEEQDERQFPFPRPPHQ EERKQEE DEDEEQQRESEESEDSELRRHKNKNPFLFGSNRFETLFKNQYGRIRVLQRFNQRSP QLQNLRDYRJLEFNSKPNTLLLPNHADADYL1VILNGTAILSLVNNDDRDSYRLQSG DALRVPSGTTYYVVNPDN ENLRLITLAIPV KPGRFESFFLSSTEAQQSYLQGFSR

N1LEASYDTKFEEINKVLFSREEGQQQGEQRLQESV1VEISKEQIRALSKRAKSSSRK TIS SEDKPFNLRSRDPIYSNKLGKFFEITPEKNPQLRDLDIFLSIVDMNEGALLLPHFN SKAIVILVINEGDANIELVGLKEQQQEQQQEEQPLEVRKYRAELSEQDIFVIPAGYP VVV ATSNLNFFAIG1NAEN QR FLAGSQDNVISQIPSQVQELAFPGSAQAVEKL LKNQRESYFVDAQPKKKEEGNKGRKGPLSSILRAFY SEQ ID NO: 68 Gly m 5 Glyma20g28660.1 BLAST 606aa

MMRARFPLLLLGLVFLASVSVSFGIAYWEKENPKHNKCLQSCNSERDSYRNQACH ARCNLLKVEKEECEEGEIPRPRPRPQHPEREPQQPGEKEEDEDEQPRPIPFPRPQPRQ EEEHEQREEQEWPRKEEKRGEKGSEEEDEDEDEEQDERQFPFPRPPHQKEERKQEE DEDEEQQRESEESEDSELRRHKNKNPFLFGSNRFETLFKNQYGRIRVLQRFNQRSP QLQNLRDYRILEFNSKPNTLLLPNHADADYLIVILNGTAILSLVNNDDRDSYRLQSG DALRVPSGTTYYVVNPDN ENLRLITLAIPVNKPGRFESFFLSSTEAQQSYLQGFSR NILEA S YDTKFEEINKVLF SREEGQQQGEQRLQES VI VEI SKEQIRAL SKRAKS S SRK TISSEDKPFNLRSRDPIYSNKLGKFFEITPEKNPQLRDLDIFLSIVDMNEGALLLPHFN SKAIVILVINEGDANIELVGLKEQQQEQQQEEQPLEVRKYRAELSEQDIFVIPAGYP VVVNATSNLNFFAIGINAENNQRNFLAGSQDNVISQIPSQVQELAFPGSAQAVEKL LKNQRESYFVDAQPKKKEEGNKGRKGPLSSILRAFY

SEQ ID NO: 69 Gly m 5 Glyma20g28650.2 BLAST 543aa

MMR ARFPLLLLGL VFL A S V S V SFGI A Y WEKENPKHNKCLQ SCN SERDS YRNQ ACH ARCNLLKVEKEECEEGEIPRPRPRPQHPEREPQQPGEKEEDEDEQPRPIPFPRPQPRQ EEEHEQREEQEWPRKEEKRGEKGSEEEDEDEDEEQDERQFPFPRPPHQKEERKQEE DEDEEQQRESEESEDSELRRHKNKNPFLFGSNRFETLFKNQYGRIRVLQRFNQRSP QLQNLRDYRILEFNSKPNTLLLPNHADADYLIVILNGTAILSLVNNDDRDSYRLQSG DALRVPSGTTYYVVNPDNNENLRLITLAIPVNKPGRFESFFLSSTEAQQSYLQGFSR NILEASYDTKFEEINKVLFSREEGQQQGEQRLQESVIVEISKEQIRALSKRAKSSSRK TISSEDKPFNLRSRDPIYSNKLGKFFEITPEKNPQLRDLDIFLSIVDMNEGALLLPHFN SKAIVILVINEGDANIELVGLKEQQQEQQQEEQPLEVRKYRAELSEQDIFVIPAGYP VVVNATSNLNFFAIGINAENNQRNFLAGI SEQ ID NO: 70 Gly m 5 Ping AAA33947 218aa

SKRAKSSSRKTISSEDKPFNLGSRDPIYSKKLGKFFEITPEKNPQLRDLDIFLSIVDMN EGALLLPHFNSKAIVILVINEGDANIELVGLKEQQQEQQQEEQPLEVRKYRAELSEQ DIFVIPAGYPVVVNATSNLNFFAIGINAENNQRNFLAGSQDNVISQIPSQVQELAFPG SAQAVEKLLKNQRESYFVDAQPNEKEEGNKGRKGPLSSILRAFY

SEQ ID NO: 71 Gly m 5 NP 001237316 BLAST 621 aa

MMRARFPLLLLGVVFLASVSVSFGIAYWEKQNPSHNKCLRSCNSEKDSYRNQACH ARCNLLKVEEEEECEEGQIPRPRPQHPERERQQHGEKEEDEGEQPRPFPFPRPRQPR QEGEHEQKEEHEWHRKEE HGGKGSEEEQDGREHPRPHQPHQKEEEKHEWQHK QEKHQGKESEEEEEDQDEDEEQDKESQESEGSESQREPRRH NKNPFHFNSKRFQT LFKNQYGHVRVLQRFN RSQQLQNLRDYRILEFNSKPNTLLLPHHADADYLIVILN GTAILTLVNNDDRDSYNLQSGDALRVPAGTTYYVVNPDNDENLRMITLA1PVNKP GRFESFFLSSTQAQQSYLQGFSKNILEASYDTKFEEINKVLFGREEGQQQGEERLQE SVIVEISKKQIRELSKRAKSSSRKTISSEDKPFNLRSRDPIYSNKLG LFEITPEKNPQ LPvDLDVFLSVVDMNEGALFLPHFNSKAIVVLVINEGEANIELVGIKEQQQRQQQEE QPLEVRKYRAELSEQDIFVIPAGYPVVVNATSDLNFFAFGINAENNQR FLAGSKD NVISQIPSQVQELAFLGSAKDIENL1KSQSESYFVDAQPQQKEEGNKGRKGPLSSILR AFY

SEQ ID NO: 72 Gly m 5 BAE02726 BLAST 621aa

MMRARFPLLLLGVVFLASVSVSFGIAYWEKQNPSHNKCLRSCNSEKDSYRNQACH

ARCNLLKVEEEEECEEGQIPRPRPQHPERERQQHGEKEEDEGEQPRPFPFPRPRQPH

QEEEHEQKEEHEWHRKEEKHGGKGSEEEQDEREHPRPHQPHQKEEEKHEWQHKQ

EKHQGKESEEEEEDQDEDEEQDKESQESEGSESQREPRRHKNKNPFHFNSKRFQTL

FKNQYGHVRVLQRFNKRSQQLQNLRDYRILEFNSKPNTLLLPHHADADYLIVILNG

TAILTLVNNDDRDSYNLQSGDALRVPAGTTYYVVNPDNDENLRMITLAIPVNKPG

RFESFFLSSTQAQQSYLQGFSKNILEASYDTKFEEINKVLFGREEGQQQGEERLQES

VI VEI SKKQIREL SKH AKS S SRKTI S SEDKPFNLRSRDPI YSNKLGKLFEITPEKNPQL

RDLDVFLSVVDMNEGALFLPHFNSKAIVVLVINEGEANIELVGIKEQQQRQQQEEQ

PLEVRKYRAELSEQDIFVIPAGYPVVVNATSDLNFFAFGINAENNQRNFLAGSKDN

VISQIPSQVQELAFPGSAKDIENLIKSQSESYFVDAQPQQKEEGNKGRKGPLSSILRA

FY

SEQ ID NO: 73 Gly m 5 Ping AAB01374 639aa

MMRARFPLLLLG V VFLA S V S V SFGI A Y WEKQNP SHNKCLRSCN SEKD S YRNQ ACH

ARCNLLKVEEEEECEEGQIPRPRPQHPERERQQHGEKEEDEGEQPRPFPFPRPRQPH

QEEEHEQKEEHEWHRKEEKHGGKGSEEEQDEREHPRPHQPHQKEEEKHEWQHKQ

EKHQGKESEEEEEDQDEDEEQDKESQESEGSESQREPRRHKNKNPFHFNSKRFQTL

FKNQYGFTVRVLQRFNKRSQQLQNLRDYRILEFNSKPNTLLLPHFLADADYLIVILNG

TAILTLVNNDDRDSYNLQSGDALRVPAGTTFYWNPDNDENLRMIAGTTFYVVNP

DNDENLRMITLAIPVNKPGRFESFFLSSTQAQQSYLQGFSKNILEASYDTKFEEINK

V LFGREEGQQQGEERLQE S VI VEI SKKQIREL SKH AKS S SRKTI S SEDKPFNLGSRDP

IYSNKLGKLFEITQRNPQLRDLDVFLSVVDMNEGALFLPHFNSKAIVVLVINEGEA

NIELVGIKEQQQRQQQEEQPLEVRKYRAELSEQDIFVIPAGYPVMVNATSDLNFFA

FGINAEN QRNFLAGSKDNVISQIPSQVQELAFPRSAKDIENLIKSQSESYFVDAQP

QQKEEGNKGRKGPLSSILRAFY

SEQ ID NO: 74 Gly m 5 Glymal 0g39150.1 BLAST 622aa

MMRARFPLLLLGVVFLASVSVSFGIAYWEKQNPSHNKCLRSCNSEKDSYRNQACH

ARCNLLKVEEEEECEEGQIPRPRPQHPERERQQHGEKEEDEGEQPRPFPFPRPRQPH

QEEEHEQKEEHEWHRKEEKHGGKGSEEEQDEREHPRPHQPHQKEEEKHEWQHKQ

EKHQGKESEEEEEDQDEDEEQDKESQESEGSESQREPRRHKNKNPFHFNSKRFQTL

FKNQYGHVRVLQRFNKRSQQLQNLRDYRILEFNSKPNTLLLPHHADADYLIVILNG

TAILTLVNNDDRDSYNLQSGDALRVPAGTTYYVVNPDNDENLRMITLAIPVNKPG

RFESFFLSSTQAQQSYLQGFSKNILEASYDTKFEEINKVLFGREEGQQQGEERLQES

VIVEISKKQIRELSKHAKSSSRKTISSEDKPFNLRSRDPIYSNKLGKLFEITPEKNPQL

RDLDVFLSVVDMNEGALFLPHFNSKAIVVLVINEGEANIELVGIKEQQQRQQQEEQ

PLEVRKYRAELSEQDIFVIPAGYPVVVNATSDLNFFAFGINAENNQRNFLAGSKDN

VISQIPSQVQELAFPGSAKDIENLIKSQSESYFVDAQPQQKEEGNKGRKGPLSSILRA

FY

SEQ ID NO: 75 CNLLK SEQ ID NO: 76 EEDEDEQPRPIPFPRPQPR

SEQ ID NO: 77 EEQEWPR

SEQ ID NO: 78 QFPFPRPPHQK

SEQ ID NO: 79 ESEESEDSELR

SEQ ID NO: 80 NPFLFGSNR

SEQ ID NO: 81 FETLFK

SEQ ID NO: 82 SPQLQNLR

SEQ ID NO: 83 LQSGDALR

SEQ ID NO: 84 VPSGTTYYVVNPDNNENLR

SEQ ID NO: 85 FESFFLSSTEAQQSYLQGFSR

SEQ ID NO: 86 FEEINK

SEQ ID NO: 87 VLFSR

SEQ ID NO: 88 TISSEDKPFNLR

SEQ ID NO: 89 DPIYSNK

SEQ ID NO: 90 FFEITPEK

SEQ ID NO: 91 AIVILVINEGDANIELVGLK

SEQ ID NO: 92 EQQQEQQQEEQPLEVR

SEQ ID NO: 93 NFLAGSQDNVISQIPSQVQELAFPGSAQAVEK

SEQ ID NO: 94 ESYFVDAQPK

SEQ ID NO: 95 Gly m 6 Gl Ping CAA26723.1 495aa

MAKLVFSLCFLLFSGCCFAFSSREQPQQNECQ1QKLNALKPGNRIESEGGLIETWNP NNKPFQCAGVALSRCTLNRNALRRPSYTNGPQEIYIQQGKGIFGMIYPGCSSTFEEP QQPQQRGQSSRPQDRHQ IYNSREGDLIAVPTGVAWW YNNEDTPVVAVSIIDTN SLENQLDQMPRRFYLAGNQEQEFLKYQQEQGGHQSQKGKHQQEEENEGGSILSGF TLEFLEHAFSVDKQIAKNLQGENEGEDKGAIVTVKGGLSVIKPPTDEQQQRPQEEE EEEEDEKPQCKGKDKHCQRPRGSQSKSRRNGIDETICTMRLRHNIGQTSSPDIYNPQ AGSVTTATSLDFPALSWLRLSAGFGSLR NAMFVPHYNLNANSllYALNGRALIQV VNCNGERVFDGELQEGRVLIVPQNFVVAARSQSDNFEYVSFKTNDTPMIGTLAGA NSLLNALPEEVIQHTFNLKSQQARQIKNN PF FLVPPQESQKRAVA SEQ ID NO: 96 LVFSLCFLLFSGCCFAFSSPv

SEQ ID NO: 97 EQPQQNECQIQK

SEQ ID NO: 98 RPSYTNGPQEIYIQQGK

SEQ ID NO: 99 HQQEEENEGGSILSGFTLEFLEHAFSVDK

SEQ ID NO: 100 HCQRPR

SEQ ID NO: 101 HNIGQTSSPDIYNPQAGSVTTATSLDFPALSWLR

SEQ ID NO: 102 ALIQVVNCNGER

SEQ ID NO: 103 VFDGELQEGR

SEQ ID NO: 104 TNDTPMIGTLAGANSLLNALPEEVIQHTFNLK

SEQ ID NO: 105 NNNPFK

SEQ ID NO: 106 FLVPPQESQK

SEQ ID NO: 107 Gly m 6 G2 Ping CAA26575.1 485aa

MAKLVLSLCFLLFSGCFALREQAQQNECQIQKLNALKPGNRIESEGGFIETWNPNN KPFQCAGVALSRCTLNRNALRRPSYTNGPQEIYIQQGNGIFGMIFPGCPSTYQEPQE SQQRGRSQRPQDRHQKVHRFREGDLIAVPTGVAWWMYNNEDTPVVAVSIIDTNS LENQLDQMPRRFYLAGNQEQEFLKYQQQQQGGSQSQKGKQQEEENEGSNILSGF APEFLKEAFGVNMQIVRNLQGENEEEDSGAIVTVKGGLRVTAPAMRKPQQEEDDD DEEEQPQCVETDKGCQRQSKRSRNGIDETICTMRLRQNIGQNSSPDIYNPQAGSITT ATSLDFPALWLLKLSAQYGSLRKNAMFVPHYTLNANSIIYALNGRALVQVVNCNG ERVFDGELQEGGVLIVPQNFAVAAKSQSDNFEYVSFKTNDRPSIGNLAGANSLLNA LPEEVIQHTFNLKSQQARQVKNNNPFSFLVPPQESQRRAVA

SEQ ID NO: 108 LVLSLCFLLFSGCFALR

SEQ ID NO: 109 EQAQQNECQIQK

SEQ ID NO: 1 1 0 RPSYTNGPQE1YIQQGNGIFGMIFPGCPSTYQEPQESQQR

SEQ ID NO: 1 1 1 SQRPQDR

SEQ ID NO: 1 12 QQEEENEGSNILSGFAPEFLK

SEQ ID NO: 1 13 EAFGVNMQIVR

SEQ ID NO: 1 14 KPQQEEDDDDE EEQPQC VETDK

SEQ ID NO: 1 1 5 LSAQYGSLR SEQ ID NO: 1 16 N AMF VPHYTLN AN SII Y ALNGR

SEQ ID NO: 1 17 ALVQVV CNGER

SEQ ID NO: 118 VFDGELQEGGVLIVPQNFAVAAK

SEQ ID NO: 119 TNDRPSIGNLAGANSLLNALPEEVIQHTFNLK

SEQ ID NO: 120 NNNPFSFLVPPQESQR

Signature Peptides for Gly m 6 G3

SEQ ID NO: 121 LVLSLCFLLFSGCCFAFSFR

SEQ ID NO: 122 EQPQQNECQIQR

SEQ ID NO: 123 QQEEENEGG SIL SGF APEFLEHAF V VDR

SEQ ID NO: 124 LQGENEEEEK

SEQ ID NO: 125 GGLSVISPPTEEQQQRPEEEEKPDCDEK

SEQ ID NO: 126 HCQSQSR

SEQ ID NO: 127 LSAQFGSLR

SEQ ID NO: 128 VFDGELQEGQVLIVPQNFAVAAR

SEQ ID NO: 129 TNDRPSIGNLAGANSLLNALPEEVIQQTFNLR

SEQ ID NO: 130 Gly m 6 G4 consensus sequence

MGKPFTLSLSSLCLLLLSSACFAISSSKLNECQLNNLNALEPDHRVESEGGLIQTWN SQHPELKCAGVTVSKLTLNRNGLHLPSYSPYPRMIIIAQGKGALGVAIPGCPETFEE PQEQSNRRGSRSQKQQLQDSHQKIRHFNEGDVLVIPPGVPYWTYNTGDEPVVAISL LDTSNFNNQLDQTPRVFYLAGNPDIEYPETMQQQQQQKSHGGRKQGQHQQEEEE EGGSVLSGFSKHFLAQSFNTNEDIAEKLQSPDDERKQIVTVEGGLSVISPKWQEQQ DEDEDEDEDDEDEQ1PSHP

SEQ ID NO: 131 Gly m 6 G4 Ping CAA60533.1 563aa

MGKPFTLSLSSLCLLLLSSACFAISSSKLNECQLNNLNALEPDHRVESEGGLIQTWN SQHPELKCAGVTVSKLTLNRNGLHLPSYSPYPRMIIIAQGKGALGVAIPGCPETFEE PQEQSNRRGSRSQKQQLQDSHQKIRHFNEGDVLVIPPGVPYWTYNTGDEPVVAISL LDTSNFNNQLDQTPRVFYLAGNPDIEYPETMQQQQQQKSHGGRKQGQHQQEEEE EGGSVLSGFSKHFLAQSFNTNEDIAEKLQSPDDERKQIVTVEGGLSVISPKWQEQQ DEDEDEDEDDEDEQIPSHPPRRPSHGKREQDEDEDEDEDKPRPSRPSHGKREQDQD QDEDEDEDEDQPRKSREWRSKKTQPRRPRQEEPRERGCETRNGVEENICTLKLHE NIARPSRADFYNP AGRISTLNSLTLPALRQFQLSAQYVVLYKNG1YSPHWNLNAN SVIYVTRGQGKVRVVNCQGNAVFDGELRRGQLLVVPQNFVVAEQAGEQGFEYIV FKTHHNAVTSYLKDVFRAIPSEVLAHSYNLRQSQVSELKYEGNWGPLVNPESQQG SPRVKVA

SEQ ID NO: 132 Gly m 6 G4 UniProt Q9SB 1 1 G4 563aa

MGKPFTLSLSSLCLLLLSSACFAISSSKLNECQLNNLNALEPDHRVESEGGLIQTWN SQHPELKCAGVTVSKLTLNRNGLHLPSYSPYPRMIIIAQGKGALGVAIPGCPETFEE PQEQSNRRGSRSQKQQLQDSHQKIRHFNEGDVLVIPPGVPYWTYNTGDEPVVAISL LDTSNFNNQLDQTPRVFYLAGNPDIEYPETMQQQQQQKSHGGRKQGQHQQEEEE EGGSVLSGFSKHFLAQSFNTNEDIAEKLQSPDDERKQIVTVEGGLSVISPKWQEQQ DEDEDEDEDDEDEQIPSHPPRRPSHGKREQDEDEDEDEDKPRPSRPSQGKREQDQD QDEDEDEDEDQPRKSREWRSKKTQPRRPRQEEPRERGCETRNGVEENICTLKLHE NIARPSRADFYNPKAGRISTLNSLTLPALRQFQLSAQYVVLYKNGIYSPHWNLNAN SVIYVTRGQGKVRVVNCQGNAVFDGELRRGQLLVVPQNFVVAEQAGEQGFEYIV FKTHFINAVTSYLKDVFRAIPSEVLAHSYNLRQSQVSELKYEGNWGPLV PESQQG SPRVKVA

SEQ ID NO: 133 Gly m 6 G4 Ping CAA55977.1 517aa

MGKPFFTLSLSSLCLLLLSSACFAITSSKFNECQLNNLNALEPDHRVESEGGLIETW NSQHPELQCAGVTVSKRTLNRNGLHLPSYSPYPQMIIVVQGKGAIGF AFPGCPETFE KPQQQSSRRGSRSQQQLQDSHQKIRHFNEGDVLVIPPGVPYWTYNTGDEPVVAISL LDTSNF QLDQNPRVFYLAGNPDIEHPETMQQQQQQKSHGGRKQGQHQQQEEE GGSVLSGFSKHFLAQSFNTNEDTAEKLRSPDDERKQIVTVEGGLSVISPKWQEQED EDEDEDEEYEQTPSYPPRRPSHGKHEDDEDEDEEEDQPRPDHPPQRPSRPEQQEPR GRGCQTRNGVEENICTMKLHENIARPSRADFYNPKAGRISTLNSLTLPALRQFGLS AQYVVLYRNGIYSPHWNLNANSVIYVTRGKGRVRVV CQGNAVFDGELRRGQLL VVPQNFVVAEQGGEQGLEYVVFKTHHNAVSSYIKDVFRAIPSEVLSNSYNLGQSQ VRQLKYQGNSGPLVNP

SEQ ID NO: 134 Gly m 6 G4 Ping AAA33964.1 516aa

MGKPFFTLSLSSLCLLLLSSACFAITSSKFNECQLNNLNALEPDHRVESEGGLIETW NSQHPELQC AG VTV SKRTLNRNG SHLP S YLPYPQMII V VQGKG AIGF AFPGCPETFE KPQQQS SRRG SRSQQQLQDSHQKIRHFNEGDVLVIPLG VPY WTYN TGDEPVVAI SP LDTSNFNNQLDQNPRVFYLAGNPDIEHPETMQQQQQQKSHGGRKQGQHRQQEEE GGSVLSGFSKHFLAQSFNTNEDTAEKLRSPDDERKQIVTVEGGLSVISPKWQEQED EDEDEDEE YGRTPS YPPRRPSHGKHEDDEDEDEEEDQPRPDHPPQRPSRPEQQEPR GRGCQTRNGVEENICTMKLHENIARPSRADFYNPKAGRISTLNSLTLPALRQFGLS AQYVVLYRNGIYSPDWNLNANSVTMTRGKGRVRVVNCQGNAVFDGELRRGQLL VVPQNPAVAEQGGEQGLEYVVFKTHHNAVSSY1KDVFRVIPSEVLSNSYNLGQSQ VRQLKYQGN SGPLVNP

SEQ ID NO: 135 LNECQLNNLNALEPDHR

SEQ ID NO: 136 CAGVTVSK

SEQ ID NO: 137 LTLNR

SEQ ID NO: 138 MIIIAQGK

SEQ ID NO: 139 GALGVAIPGCPETFEEPQEQSNR SEQ ID NO: 140 QQLQDSHQK

SEQ ID NO : 141 VFYLAGNPDIEYPETMQQQQQQK

SEQ ID NO: 142 QGQHQQEEEEEGGS VLSGFSK

SEQ ID NO: 143 HFLAQSFNTNEDIAEK

SEQ ID NO: 144 QIVTVEGGLSVISPK

SEQ ID NO: 145 WQEQQDEDEDEDEDDEDEQIPSHPPR

SEQ ID NO: 146 RPSHGK

SEQ ID NO: 147 EQDEDEDEDEDKPRPSRPSQGK

SEQ ID NO: 148 QEEPR

SEQ ID NO: 149 NGVEENICTLK

SEQ ID NO: 150 LHENIARPSR

SEQ ID NO: 151 ISTLNSLTLPALR

SEQ ID NO: 152 QFQLSAQYVVLYK

SEQ ID NO: 153 NGIYSPHWNLN ANSVIYVTR

SEQ ID NO: 154 DVFR

SEQ ID NO: 155 AIPSEVLAHSYNLR

SEQ ID NO: 156 QSQVSELK

SEQ ID NO: 58 YEGNWGPLVNPESQQGSPR

Signature Peptides for Gly m 6 precursor

SEQ ID NO: 59 GALQCKPGCPETFEEPQEQSNR

SEQ ID NO: 60 LQSPDDER

Claims

CLAIMS We claim:

1. A method of selecting candidate signature peptide for quantitation of known allergen and potential allergens from a plant-based sample, comprising:

2. The method of claim 1 , wherein the quantitating step uses a column chromatography and mass spectrometry.

3. The method of claim 1 , wherein the quantitating step comprises measuring the plural of candidate signature peptides using high resolution accurate mass spectrometry (HRAM MS).

4. The method of claim 1 , wherein the quantitating step comprises calculating corresponding peak heights or peak areas of the candidate signature peptides from mass spectrometry.

5. The method of claim 1 , wherein the quantitating step comprises comparing data from high fragmentation mode and lo fragmentation mode from mass spectrometry.

6. The method of claim 1 , wherein the at least one known allergen comprises Gly m 1 , Gly m 3, Gly m 4, Gly m 5 (beta-conglycinin), Gly m 6 (Glycinin) Gl , Gly m 6 (Glycinin) G2, Gly m 6 (Glycinin) G3, Gly m 6 (Glycinin) G4, or Gly m 6 (Glycinin) precursor.

7. The method of claim 1, wherein the potential allergens comprise at least one sequence selected from the group consisting of:

(a) SEQ ID NOs 12 and 30-35 for Gly m 1 ;

(b) SEQ ID NOs 13 and 39-40 for Gly m 3;

(c) SEQ ID NOs 14 and 43-51 for Gly m 4;

(d) SEQ ID NOs 15 and 62-74 for Gly m 5;

(e) SEQ ID NOs 16 and 95 for Gly m 6 Gl ;

(f> SEQ ID NOs 17 and 107 for Gly m 6 G2;

(g) SEQ ID NO: 18 for Gly m 6 G3;

(h) SEQ ID NOs 19 and 131 -134 for Gly m 6 G4; and

(0 SEQ ID NO: 20 for Gly m 6 precursor.

8. The method of claim 1 , wherein the candidate signature peptides comprise at least one sequence selected from the group consisting of:

(a) SEQ ID NOs: 1, 36, and 37 for Gly m 1 ;

(b) SEQ ID NOs: 2 and 41 for Gly m 3;

(c) SEQ ID NOs: 52-57 for Gly m 4;

(d) SEQ ID NOs: 3 and 75-94 for Gly m 5;

(e) SEQ ID NOs: 96-106 for Gly m 6 Gl ;

(f) SEQ ID NOs: 4 and 108- 120 for Gly m 6 G2;

(g) SEQ ID NOs: 5 and 121 -129 for Gly m 6 G3;

(h) SEQ ID NOs: 135-156 and 58 for Gly m 6 G4; and

(0 SEQ ID NOs: 6, 59, and 60 for Gly m 6 precursor.

9. The method of claim 1 , wherein the plant-based sample comprises a soybean seed or part of a soybean seed.

10. A system for quantitating one or more protein of interest with known amino acid sequence in a plant-based sample, the system comprising:

(a) a high-throughput means for extracting proteins from a plant-based sample;

(c) a separation module for separating peptides in a single step;

(e) a mass spectrometry for measuring the plural of signature peptides.

1 1. The system of claim 10, wherein the separation module comprises a column chromatography.

12. The system of claim 1 1 , wherein the column chromatography comprises a liquid column chromatography.

13. The system of claim 10, wherein the mass spectrometry comprises a high resolution accurate mass spectrometry (HRAM MS).

14. The system of claim 10, wherein the selection module uses a method according to claim 1.

15. The system of claim 10, wherein the at least one known allergen comprises Gly m 1 , Gly m 3, Gly m 4, Gly m 5 (beta-conglycinin), Gly m 6 (Glycinin) G l , Gly m 6 (Glycinin) G2, Gly m 6 (Glycinm) G3, Gly m 6 (Glycinin) G4, or Gly m 6 (Glycinin) precursor.

16. The system of claim 10, wherein the potential allergens comprise at least one sequence selected from the group consisting of:

(a) SEQ ID NOs 12 and 30-35 for Gly m 1 ;

(b) SEQ ID NOs 13 and 39-40 for Gly m 3;

(c) SEQ ID NOs 14 and 43-51 for Gly m 4;

(d) SEQ ID NOs 1 5 and 62-74 for Gly m 5;

(e) SEQ ID NOs 16 and 95 for Gly m 6 G l ; (f) SEQ ID NOs: 17 and 107 for Gly m 6 G2;

(g) SEQ ID NO: 18 for Gly m 6 G3;

(h) SEQ ID NOs: 19 and 131-134 for Gly m 6 G4; and

(i) SEQ ID NO: 20 for Gly m 6 precursor.

17. The system of claim 10, wherein the signature peptides comprise at least one sequence selected from the group consisting of:

(a) SEQ ID NOs: 1 , 36, and 37 for Gly m 1 ;

(b) SEQ ID NOs: 2 and 41 for Gly m 3;

(c) SEQ ID NOs: 52-57 for Gly m 4;

(d) SEQ ID NOs: 3 and 75-94 for Gly m 5;

(e) SEQ ID NOs: 96-106 for Gly m 6 Gl ;

(f) SEQ ID NOs: 4 and 108-120 for Gly m 6 G2;

(g) SEQ ID NOs: 5 and 121-129 for Gly m 6 G3;

(h) SEQ ID NOs: 135-156 and 58 for Gly m 6 G4; and

(0 SEQ ID NOs: 6, 59, and 60 for Gly m 6 precursor..

18. The system of claim 10, wherein the plant-based sample comprises a soybean seed or part of a soybean seed.

19. A high-throughput method of quantitating at least one allergen with known amino acid sequence and homologous potential allergens in a plant-based sample, comprising using the system of claim 10.