Abstract:
Disclosed are methods for determining the presence of one or more proteins in a sample, the methods comprising: enzymatically digesting the sample with a protease activity to generate a plurality of proteolytic peptides; separating the plurality of proteolytic peptides using liquid chromatography; performing mass spectrometry on the separated plurality of peptides; and wherein a protein is present in the sample when three or more target peptides for the protein are present among the proteolytic peptides; and wherein the target peptides are selected from the groups consisting of SEQ ID NOS:6-8, SEQ ID NOS:9-11, SEQ ID NOS:12-14, SEQ ID NOS:15-17, and SEQ ID NOS:18-20. In embodiments, a known quantity of a standard peptide may be added to the proteolytic peptides.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 62/359,310, filed Jul. 7, 2016, the disclosure of which is hereby incorporated herein in its entirety by this reference. 
     
    
     TECHNICAL FIELD 
       [0002]    The present application relates generally to biotechnology. More specifically, embodiments of the application relate to the detection of specific proteins in a sample through the use of liquid chromatography and mass spectrometry. 
       BACKGROUND 
       [0003]    In various fields ranging from fundamental biology to clinical diagnostic and public health surveillance, the specific and accurate identification and quantification of proteins in complex biological samples remains a recurrent and challenging problem. For many protein biomarkers, this problem has been solved by immunological techniques. However, the success of immunological approaches relies on the heavy duty production and validation of high specificity and high affinity antibodies. Although recent efforts are being made to design antibodies arrays, the adaptation of immunological methods to multiplexed analyses remains limited. Indeed, the simultaneous optimization of several protein assays is hardly ever possible. Alternatively, the power of mass spectrometry (MS)-based proteomics can be harnessed to determine the presence of specific proteins in a sample. 
       BRIEF SUMMARY 
       [0004]    Embodiments of the invention relate to methods for identifying and/or quantifying a target polypeptide or target polypeptides in a sample comprising the steps of:
       providing a sample to be analyzed;   treating the sample with a protease activity to generate a plurality of proteolytic peptides;   separating the proteolytic peptides via liquid chromatography;   analyzing the separated proteolytic peptides by mass spectrometry (MS); and/or   analyzing the results of the MS to determine the presence of particular target fragments.       
 
         [0010]    In embodiments, a known quantity of an internal standard spike may be added to the sample, thereby generating a spiked sample. In further embodiments, a protein may be identified as being present in the sample where three or more target fragments specific for a particular protein are found among the proteolytic fragments. 
         [0011]    These and other aspects of the disclosure will become apparent to the skilled artisan in view of the teachings contained herein. 
     
    
     DETAILED DESCRIPTION 
       [0012]    Embodiments of the invention include methods of detecting the presence of a particular polypeptide in a sample. Examples of proteins that can be detected using the methods described herein include, but are not limited to, α-S1-Casein, β-Lactoglobulin, Vicilin, Glutelin, and Glycinin G1 (SEQ ID NOS: 1-5 of the Sequence Listing incorporated herein, respectively). 
         [0013]    The term “target fragment” refers to a specific polypeptide obtained after proteolysis of a polypeptide to be detected, which is a fragment of a larger protein. Examples of target fragments include, but are not limited to, SEQ ID NOS:6-20. 
         [0014]    As used herein, a “protease activity” is an activity that cleaves amide bonds in a polypeptide. The activity may be implemented by an enzyme such as a protease or by a chemical agent. Suitable proteases include, but are not limited to one or more of serine proteases (e.g., such as trypsin, hepsin, SCCE, TADG12, TADG14); metalloproteases (e.g., such as PUMP-1); chymotrypsin; cathepsin; pepsin, elastase; pronase; Arg-C; Asp-N; Glu-C; Lys-C; carboxypeptidases A, B, and/or C; dispase; thermolysin; cysteine proteases such as gingipains, and the like. Proteases may be isolated from cells or obtained through recombinant techniques. Chemical agents with a protease activity such as CNBr can also be used. In embodiments, the sample may be subjected to the protease activity until essentially all cleavage sites have been acted upon. 
         [0015]    The method described herein may be used in a large variety of fields; such as proteomics, detection of biomarkers in biological samples, quality controls in the manufacture of vaccines and other bioproducts, biological and health hazard controls, food, detection of specific ingredients in foods and/or raw materials, and/or water controls. 
         [0016]    Typically, the protein to be detected may be a biomarker, a protein or a fragment thereof which is physically, physiologically, or pathologically present in a sample, a bacterial protein, a viral protein, a plant protein, a yeast protein, a mold protein, a fungal protein, an animal protein or a toxin. 
         [0017]    The size of the target fragment may be any size as long as the presence of the target fragment is detectable by the methods described herein. For example, target fragments, may be about 10, 15, 20, 25, 30, 35, 40, or 50 polypeptides in length. 
         [0018]    Examples of samples on which the methods may be performed are foods, food ingredients, nutraceuticals, biological fluids (for example, but not limited to, blood, serum, plasma, cerebrospinal fluid, urine, saliva, and lachrymal fluid), tissue and cells homogenates, cell culture supernantants, water, biocollection fluids and any biochemical fraction derived from the above materials. Biocollection fluids are fluids which are used for collecting particles which may be present in air or gas samples. 
         [0019]    Examples of foods and food ingredients include, but are not limited to, cow&#39;s milk, pea, rice, soy, and wheat. 
         [0020]    The method described herein may also allow the simultaneous detection of more than one target fragment. In specific embodiments, the three or more different target fragments may be used in combination to detect the presence of a particular polypeptide in the sample. Multiplex detection of target fragments may also be performed including the detection of one or more proteins via one or more sets of target fragments. 
         [0021]    In certain embodiments, a known quantity of standard may be added to the proteolytic fragments before analysis. For example, a known quantity of β-Casomorphin 1-4 may be added to the proteolytic fragments as an internal control. Examples of known quantities of a standard include, but are not limited to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 1000, 1500, 2000, 2500, 3000, 3500, and 4000 ng/mL of the standard. 
         [0022]    In embodiments, the proteolytic peptides may be separated by chromatography prior to analysis with mass spectrometry. Examples of chromatography include, but are not limited to, liquid, affinity, ion exchange, size exclusion, expanded bed adsorption, reversed phase, two-dimensional, simulated moving-bed, fast protein liquid, countercurrent, and chiral chromatography. In certain embodiments, the chromatography may be high-performance liquid chromatography. 
         [0023]    Examples of stationary phases used in liquid chromatography include, but are not limited to, alkyl, polar, amide, phenyl, chiral, and ion pairing phases. 
         [0024]    In further embodiments, the presence of a target peptide among the proteolytic fragments may be detected by mass spectrometry. Generally, mass spectrometry ionizes chemical species and sorts the ions based on their mass to charge ratio. In this way, specific chemical species (e.g. target fragments) may be detected in a complex sample. 
         [0025]    The disclosed methods will become further apparent to the skilled artisan in view of the following examples. 
       EXAMPLES 
     Example 1: Peptide Selection 
       [0026]    To qualitatively identify a protein source, unique peptides (relative to other peptides from other proteins in the assay) that act as markers for specific proteins originating from the source species were chosen. To identify these unique marker peptides, signature protein(s) from each source were identified via literature search. The protein in cow&#39;s milk is approximately 80% casein protein and 20% whey protein [1]. These two fractions can be purified separately yielding distinct raw materials. Therefore a signature protein for both the casein and whey fractions was needed. The proteins chosen were α-S1-Casein (Cow Milk, casein fraction) [1,2], β-Lactoglobulin (Cow Milk, whey fraction) [1,2], Vicilin (Pea) [3,4], Glutelin (Rice) [3,5] and Glycinin G1 (Soy) [3,6]. 
         [0027]    An in silico digestion was performed on the signature proteins to generate tryptic peptides [7]. As high level plant proteins tend to belong to related seed storage protein families they are likely to have homologous amino acid sequences. A Basic Local Alignment Search Tool (BLAST) [8] search was performed to establish peptide specificity at this stage of development. The specific peptides were then analyzed by LC/MS/MS. 
       Example 2: Sample Preparation and Testing 
       [0028]    Sample Preparation 
         [0029]    Samples were thoroughly mixed before weighing. 500 mg of sample was transferred to a 15 mL centrifuge tube. 10 mL of the extraction buffer (300.3 g of Urea (MW=60.06) and 6.057 g Tris base (MW=121.14) were transferred to a 1 liter graduated cylinder and combined with ultrapure water to form 1 Liter of solution) was added and the resulting solution vortexed for 10-60 seconds until the sample material was a homogenous slurry. The slurry was then sonicated for 60 minutes. 
         [0030]    After sonication the tubes were spun down for 10 minutes at 4000 g to pellet remaining solids. 6 mL of supernatant was transferred by pipette to a new 15 mL conical centrifuge tube and 200 μL of the thawed reducing solution (1.697 g of dithiotreitol (DTT, MW=154.253) in 11 mL ultrapure water) was added. The resulting solution was vortexed to mix and heated for 30 minutes in a 55° C. water bath. After cooling to room temperature, 2.0 mL of the freshly prepared alkylating solution (185 mg of iodoacetamide (MW=184.96) in 2 mL of ultrapure water) was added to the samples. The samples were then mixed by vortexing and stored with the tube protected from light for 30 minutes at room temperature. 
         [0031]    Following alkylation, 6 mL of digestion buffer (3.953 g of ammonium bicarbonate (MW=79.06) in 400 mL ultrapure water) was added to each sample. After carefully mixing by inverting tube multiple times. 400 μL of the thawed trypsin working solution (500 μg/mL in digestion buffer) (200 μg trypsin per sample) was added and mixed by carefully inverting tube multiple times. The samples are then incubated overnight in an incubator at 37° C. with times at start and finish of incubation recorded. 
         [0032]    10 mL of each sample were transferred by pipette to a new 15 mL conical centrifuge tube and 417 μL formic acid added and mixed by inverting the tube multiple times to quench the samples. The samples were then centrifuged for 10 minutes at 4000 g to pellet any precipitate. The resulting supernatant was then filtered using a disposable syringe and a 0.22 or 0.45 μm nylon syringe filter into a new 15 mL conical centrifuge tube. 
         [0033]    The sample was then diluted 1:10 with 0.1% formic acid and internal standard spike solution (2000 ng/mL B-Casomorphin 1-4 (YPFP) in PBS). Example: Add 100 μL of sample to autosampler vial, add 100 μL of internal standard spike solution and dilute with 800 μL 0.1% formic acid. 
         [0034]    Measurement of Target Fragments 
         [0035]    Approximately 5 μL of sample was injected into HPLC column linked to mass spectrometer for analysis. 
         [0036]    Liquid Chromatoraphic Conditions (Shimadzu Prominence High-performance Liquid Chromatography (HPLC) System) 
         [0037]    Column (Phenomenex HPLC column (C18, 150×2 mm, Synergi 4μ Hydro 80 Å, Part Number 00F-4375-B0)) Parameters (Table 1) 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 HPLC Column 
                 Luna 3 μm, 150 × 2 mm C18, 200 Å 
               
             
          
           
               
                 Column Temperature 
                 30° 
                 C. 
               
               
                 Recommended Injection Volume 
                 5 
                 μL 
               
               
                 Flow Rate 
                 0.3 
                 mL/min 
               
               
                   
               
             
          
         
       
     
         [0038]    Pump Gradient (Table 2) 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Time (min) 
                 A (%) 
                 B (%) 
                 Curve 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 0.0 
                 85 
                 15 
                 0 
               
               
                   
                 0.8 
                 85 
                 15 
                 0 
               
               
                   
                 5.1 
                 50 
                 50 
                 0 
               
               
                   
                 6.4 
                 2 
                 98 
                 0 
               
               
                   
                 7.3 
                 2 
                 98 
                 0 
               
               
                   
                 7.5 
                 85 
                 15 
                 0 
               
               
                   
                 10 
                 85 
                 15 
                 0 
               
               
                   
                   
               
             
          
         
       
     
         [0039]    Valco Valve 2-Position Schedule 
         [0040]    Position A: Flow from column diverted to waste 
         [0041]    Position B: Flow from column sent to MS for analysis 
         [0042]    Schedule (Table 3) 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Time (min) 
                 Position (A/B) 
                 Destination 
               
               
                   
                   
               
             
             
               
                   
                 0 
                 A 
                 Waste 
               
               
                   
                 1 
                 B 
                 MS analysis 
               
               
                   
                 9 
                 A 
                 Waste 
               
               
                   
                   
               
             
          
         
       
     
         [0043]    Mass Spectrometer Conditions (SCIEX 4000 Q TRAP Triple Quadrupole Mass Spectrometer Detector) 
         [0044]    Typical Instrument Settings (Table 4) 
         [0000]    
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                   
               
             
             
               
                 Fixed 
               
             
          
           
               
                   
                 Ion Transfer Voltage 
                 IS 
                 5500 
               
               
                   
                 Probe Temperature 
                 TEM 
                 500 
               
               
                   
                 Interface Heater 
                 ihe 
                 ON 
               
             
          
           
               
                 Typical 
               
             
          
           
               
                   
                 Curtain Gas 
                 CUR 
                 20 
               
               
                   
                 Ion Source Gas 1 
                 GS1 
                 40 
               
               
                   
                 Ion Source Gas 1 
                 GS2 
                 50 
               
               
                   
                 Collision Gas 
                 CAD 
                 High 
               
               
                   
                   
               
             
          
         
       
     
       Example 3: Method Reporting Limit (MRL) Testing 
       [0045]    The MRL for each marker peptide was examined in protein free matrices spiked with relevant protein raw materials at 100, 500 and 1000 ppm. These composite spikes were then analyzed to assess the MRL for each marker peptide. The criteria for MRL acceptance was that all three peptides must be present at least one spike level and that the highest blank peak area cannot exceed 20% of the MRL peak area. 
         [0046]    A composite sample of 12 separate protein samples was prepared as outlined in Table 5. The protein percent, as determined by Kjeldahl, for each individual protein sample was used to ensure that each protein source was at the same level in the final composite. This protein mix sample was used for precision testing and the creation of spiked samples used for MRL evaluation. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Composite protein mix of samples used for MRL testing. 
               
             
          
           
               
                   
                   
                 Protein % 
                 Weight 
                 Protein 
                 Combined 
               
               
                 Source 
                 SID 
                 (Kjeldahl) 
                 (g) 
                 % 
                 % 
               
               
                   
               
             
          
           
               
                 Cow Milk 
                 405984 
                 80.37% 
                 17.416 
                 7.03% 
                 21.1% 
               
               
                   
                 410058 
                 80.95% 
                 17.279 
                 7.03% 
               
               
                   
                 410057 
                 79.89% 
                 17.511 
                 7.03% 
               
               
                 Pea 
                 409386 
                 85.85% 
                 16.296 
                 7.03% 
                 21.1% 
               
               
                   
                 404987 
                 79.25% 
                 17.661 
                 7.03% 
               
               
                   
                 407133 
                 80.17% 
                 17.452 
                 7.03% 
               
               
                 Rice 
                 408894 
                 83.71% 
                 16.729 
                 7.04% 
                 21.1% 
               
               
                   
                 409338 
                 85.25% 
                 16.421 
                 7.03% 
               
               
                   
                 405915 
                 85.84% 
                 16.306 
                 7.03% 
               
               
                 Soy 
                 405663 
                 92.10% 
                 15.204 
                 7.04% 
                 21.1% 
               
               
                   
                 409547 
                 90.93% 
                 15.403 
                 7.04% 
               
               
                   
                 395810 
                 91.18% 
                 15.361 
                 7.04% 
               
               
                 Total 
                   
                   
                 199.039 
                 84.39% 
               
               
                   
               
             
          
         
       
     
         [0047]    For MRL evaluation and selectivity testing negative control matrices, either a protein free raw material (pure BCAAs, Branched chain amino acids) or protein free finished good matrix (mix of BCAA raw and a finished good), were used. To evaluate the MRL, the composite protein mix was spiked into negative control matrices as detailed in Table 6. The unspiked negative control matrices were used for selectivity testing 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Spiked protein free negative control matrices used for MRL testing 
               
             
          
           
               
                   
                 Negative Control 
                   
                   
               
               
                   
                 Matrix 
                 Spike 
                 Concentration 
               
             
          
           
               
                 Name 
                 Material 
                 (g) 
                 Material 
                 (g) 
                 % Protein 
                 ppm 
               
               
                   
               
             
          
           
               
                 RM_1_20000 
                 BCAA, 
                 8.964 
                 Protein Mix 
                 0.9364 
                 1.995% 
                 19954 
               
               
                 RM_1_1000 
                 410640 
                 9.538 
                 RM_1_20000 
                 0.5016 
                 0.100% 
                 997 
               
               
                 RM_1_500 
                   
                 9.731 
                 RM_1_20000 
                 0.2506 
                 0.050% 
                 501 
               
               
                 RM_1_100 
                   
                 9.066 
                 RM_1_1000 
                 1.006 
                 0.010% 
                 100 
               
               
                 RM_2_20000 
                 BCAA, 
                 9.024 
                 Protein Mix 
                 0.9473 
                 2.004% 
                 20043 
               
               
                 RM_2_1000 
                 410641 
                 9.493 
                 RM_2_20000 
                 0.5006 
                 0.100% 
                 1004 
               
               
                 RM_2_500 
                   
                 9.785 
                 RM_2_20000 
                 0.2490 
                 0.050% 
                 497 
               
               
                 RM_2_100 
                   
                 9.002 
                 RM_2_1000 
                 1.0010 
                 0.010% 
                 100 
               
               
                 FG_1_20000 
                 BCAA, 
                 9.049 
                 Protein Mix 
                 0.9470 
                 1.999% 
                 19987 
               
               
                 FG_1_1000 
                 410642 
                 9.520 
                 FG_1_20000 
                 0.5012 
                 0.100% 
                 1000 
               
               
                 FG_1_500 
                 (20.132 g) 
                 9.754 
                 FG_1_20000 
                 0.2511 
                 0.050% 
                 502 
               
               
                 FG_1_100 
                 FG, 
                 9.014 
                 FG_1_1000 
                 0.9985 
                 0.010% 
                 100 
               
               
                   
                 409016 
               
               
                   
                 20.124 g) 
               
               
                 FG_2_20000 
                 BCAA, 
                 9.015 
                 Protein Mix 
                 0.9457 
                 2.003% 
                 20030 
               
               
                 FG_2_1000 
                 410642 
                 9.541 
                 FG_2_20000 
                 0.5016 
                 0.100% 
                 1000 
               
               
                 FG_2_500 
                 (20.150 g) 
                 9.765 
                 FG_2_20000 
                 0.2507 
                 0.050% 
                 501 
               
               
                 FG_2_100 
                 FG, 
                 9.020 
                 FG_2_1000 
                 0.9997 
                 0.010% 
                 100 
               
               
                   
                 409101 
               
               
                   
                 (20.131 g) 
               
               
                   
               
             
          
         
       
     
         [0048]    The MRL for each marker peptide was examined in raw material (RM) and finished good (FG) negative control matrices, spiked with protein at 100, 500 and 1000 ppm (see Table 6). During MRL testing, a solution blank was injected after each sample. The peak area of marker peptides in blank injections was tracked and the highest blank peak area was used during the MRL assessment. The criteria for acceptance for each protein source were that all three peptides must be present and that the highest blank peak area cannot exceed 20% of the lowest qualifying MRL peak area. The results for MRL testing are shown in Table 7. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Observed ppm MRLs for marker peptides. Spiked negative 
               
               
                 control matrices were tested and the lowest concentration 
               
               
                 spike levels for each marker peptide which met the criteria for 
               
               
                 acceptance is shown. Samples are described in Table 6. 
               
             
          
           
               
                   
                 Cow Milk - 
                 Cow Milk - 
                   
                   
                   
               
               
                   
                 Casein 
                 Whey 
                 Rice 
                 Pea 
                 Soy 
               
             
          
           
               
                 Sample 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
             
          
           
               
                 RM-1 
                 1000 
                 1000 
                 500 
                 1000 
                 500 
                 500 
                 500 
                 100 
                 500 
                 100 
                 100 
                 100 
                 500 
                 100 
                 100 
               
               
                 RM-2 
                 1000 
                 1000 
                 500 
                 500 
                 500 
                 500 
                 100 
                 100 
                 ND 
                 100 
                 100 
                 100 
                 1000 
                 100 
                 500 
               
               
                 FG-1 
                 1000 
                 ND 
                 500 
                 500 
                 500 
                 500 
                 100 
                 100 
                 500 
                 100 
                 100 
                 100 
                 1000 
                 100 
                 100 
               
               
                 FG-2 
                 1000 
                 1000 
                 500 
                 500 
                 500 
                 500 
                 100 
                 100 
                 500 
                 100 
                 100 
                 500 
                 1000 
                 100 
                 100 
               
               
                   
               
             
          
         
       
     
         [0049]    These results indicate that the marker peptides exhibit different MRLs. In all protein free matrices, more than one peptide was assigned to each of the ppm levels tested. While further testing might identify an exact MRL, for our purposes this semiquantitative approach is sufficient as it is a conservative measure of where the lowest concentration limit can be detected. For testing, on a per assay basis the MRL will be examined using the three spike levels in the appropriate protein free matrix. This necessitates the creation of laboratory control samples (LCS) that can be used on an ongoing basis. From this testing, RM-1 and FG-2 appear appropriate for use as LCS as MRLs can be assigned for each of the marker peptides. For samples in which MRL could not be assigned for all marker peptides (Cow Milk-Casein Peptide 2 in FG-1 and Rice Peptide 3 in RM-2) these samples are deemed inappropriate for use as LCS. A complete tabulation of the MRL results including blanks are contained within Table 8. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 MRL Testing, Tabulated Data 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Cow Milk-Casein 
                 Cow Milk-Whey 
               
             
          
           
               
                 Sample Name 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
               
                 DMSO Blank 
                 45800 
                 29500 
                 5270 
                 455 
                 1040 
                 219 
               
               
                 0.1% FA Blank 
                 9340 
                 3910 
                 666 
                 33 
                 0 
                 211 
               
               
                 BCAA_1_400640 
                 10300 
                 5040 
                 812 
                 0 
                 0 
                 195 
               
               
                 0.1% FA Blank 
                 9970 
                 4750 
                 552 
                 0 
                 146 
                 127 
               
               
                 BCAA_2_400641 
                 11900 
                 5290 
                 1100 
                 33 
                 341 
                 244 
               
               
                 0.1% FA Blank 
                 8690 
                 5690 
                 449 
                 33 
                 244 
                 130 
               
               
                 BCAA_400642_C4_409016 
                 11700 
                 3610 
                 717 
                 0 
                 211 
                 0 
               
               
                 0.1% FA Blank 
                 11100 
                 5650 
                 725 
                 0 
                 130 
                 114 
               
               
                 BCAA_400642_C4_409101 
                 11200 
                 4430 
                 541 
                 0 
                 195 
                 244 
               
               
                 0.1% FA Blank 
                 10800 
                 5790 
                 552 
                 33 
                 179 
                 141 
               
               
                 DMSO Blank 
                 14100 
                 12700 
                 1860 
                 308 
                 179 
                 276 
               
               
                 0.1% FA Blank 
                 11100 
                 5000 
                 698 
                 0 
                 114 
                 195 
               
               
                 RM_1_100 
                 19300 
                 12900 
                 2090 
                 195 
                 357 
                 520 
               
               
                 0.1% FA Blank 
                 11600 
                 6230 
                 612 
                 65 
                 179 
                 195 
               
               
                 RM_2_100 
                 18200 
                 12300 
                 1610 
                 130 
                 211 
                 695 
               
               
                 0.1% FA Blank 
                 13300 
                 6000 
                 831 
                 33 
                 114 
                 260 
               
               
                 FG_1_100 
                 20700 
                 12000 
                 1610 
                 49 
                 438 
                 641 
               
               
                 0.1% FA Blank 
                 12800 
                 4770 
                 649 
                 65 
                 244 
                 276 
               
               
                 FG_2_100 
                 20100 
                 8370 
                 1540 
                 179 
                 419 
                 335 
               
               
                 0.1% FA Blank 
                 15400 
                 6260 
                 641 
                 65 
                 244 
                 276 
               
               
                 DMSO Blank 
                 17900 
                 12000 
                 1740 
                 325 
                 308 
                 127 
               
               
                 0.1% FA Blank 
                 13900 
                 5360 
                 568 
                 49 
                 162 
                 71 
               
               
                 RM_1_500 
                 69100 
                 37800 
                 5690 
                 341 
                 1830 
                 2760 
               
               
                 0.1% FA Blank 
                 13000 
                 6660 
                 744 
                 49 
                 192 
                 146 
               
               
                 RM_2_500 
                 63700 
                 41000 
                 4780 
                 649 
                 1630 
                 2060 
               
               
                 0.1% FA Blank 
                 10200 
                 7820 
                 392 
                 0 
                 81 
                 244 
               
               
                 FG_1_500 
                 54900 
                 35500 
                 6220 
                 503 
                 1590 
                 1720 
               
               
                 0.1% FA Blank 
                 13100 
                 9650 
                 503 
                 97 
                 81 
                 179 
               
               
                 FG_2_500 
                 80700 
                 43200 
                 6200 
                 552 
                 1990 
                 2460 
               
               
                 0.1% FA Blank 
                 14100 
                 10100 
                 587 
                 81 
                 97 
                 230 
               
               
                 DMSO Blank 
                 15400 
                 17100 
                 1620 
                 243 
                 227 
                 198 
               
               
                 0.1% FA Blank 
                 11300 
                 8680 
                 579 
                 81 
                 0 
                 227 
               
               
                 RM_1_1000 
                 116000 
                 67800 
                 10800 
                 1100 
                 2670 
                 3850 
               
               
                 0.1% FA Blank 
                 12400 
                 9760 
                 890 
                 0 
                 195 
                 143 
               
               
                 RM_2_1000 
                 133000 
                 77700 
                 12500 
                 974 
                 3190 
                 4330 
               
               
                 0.1% FA Blank 
                 11600 
                 7470 
                 633 
                 33 
                 222 
                 195 
               
               
                 FG_1_1000 
                 112000 
                 46100 
                 11000 
                 1280 
                 3570 
                 4470 
               
               
                 0.1% FA Blank 
                 13200 
                 11900 
                 747 
                 0 
                 130 
                 141 
               
               
                 FG_2_1000 
                 107000 
                 68100 
                 10200 
                 1010 
                 2660 
                 3340 
               
               
                 0.1% FA Blank 
                 16500 
                 9400 
                 679 
                 81 
                 162 
                 114 
               
               
                 DMSO Blank 
                 15600 
                 14000 
                 1510 
                 195 
                 179 
                 227 
               
               
                 0.1% FA Blank 
                 12600 
                 11900 
                 673 
                 64.90 
                 162 
                 130 
               
               
                   
               
             
          
           
               
                   
                 Rice 
                 Pea 
                 Soy 
               
             
          
           
               
                 Sample Name 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
               
                 DMSO Blank 
                 130 
                 0 
                 0 
                 49 
                 227 
                 0 
                 211 
                 192 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 BCAA_1_400640 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 BCAA_2_400641 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 114 
                 0 
                 0 
               
               
                 BCAA_400642_C4_409016 
                 0 
                 0 
                 0 
                 33 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 33 
                 0 
               
               
                 BCAA_400642_C4_409101 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 DMSO Blank 
                 0 
                 0 
                 0 
                 97 
                 0 
                 0 
                 438 
                 114 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 RM_1_100 
                 0 
                 584 
                 0 
                 552 
                 471 
                 114 
                 114 
                 601 
                 81 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 33 
                 0 
               
               
                 RM_2_100 
                 146 
                 720 
                 0 
                 536 
                 417 
                 162 
                 97 
                 682 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 FG_1_100 
                 162 
                 882 
                 0 
                 195 
                 487 
                 81 
                 81 
                 390 
                 97 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 FG_2_100 
                 81 
                 855 
                 0 
                 244 
                 244 
                 0 
                 0 
                 341 
                 65 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 33 
                 0 
               
               
                 DMSO Blank 
                 0 
                 0 
                 0 
                 97 
                 0 
                 0 
                 257 
                 114 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 RM_1_500 
                 509 
                 4740 
                 97 
                 2770 
                 1850 
                 520 
                 698 
                 4160 
                 771 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 RM_2_500 
                 774 
                 3620 
                 0 
                 2200 
                 2790 
                 749 
                 514 
                 2670 
                 520 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 FG_1_500 
                 552 
                 3920 
                 97 
                 796 
                 1670 
                 487 
                 179 
                 1300 
                 487 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 FG_2_500 
                 511 
                 5540 
                 49 
                 1610 
                 2020 
                 503 
                 438 
                 2730 
                 904 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 DMSO Blank 
                 0 
                 0 
                 0 
                 195 
                 0 
                 0 
                 227 
                 81 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 65 
                 33 
                 0 
               
               
                 RM_1_1000 
                 1400 
                 9840 
                 130 
                 4520 
                 5390 
                 1400 
                 1720 
                 7040 
                 1530 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 114 
                 0 
                 0 
               
               
                 RM_2_1000 
                 1650 
                 6280 
                 0 
                 4830 
                 3850 
                 1460 
                 1040 
                 6870 
                 1460 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 FG_1_1000 
                 1050 
                 8120 
                 81 
                 2400 
                 3730 
                 1410 
                 852 
                 4570 
                 1310 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 FG_2_1000 
                 863 
                 8470 
                 130 
                 2480 
                 3060 
                 1380 
                 1340 
                 6030 
                 1130 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 DMSO Blank 
                 0 
                 0 
                 0 
                 127 
                 0 
                 81 
                 179 
                 97 
                 0 
               
               
                 0.1% FA Blank 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 32.5 
                 0 
               
               
                   
               
             
          
         
       
     
       Example 3: Selectivity 
       [0050]    The following samples were used to validate selectivity, specificity, precision, and reinjection reproducibility (Table 9). 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
               
               
                 Sample 
                   
                   
                 Protein % 
               
               
                 ID 
                 Species (source) 
                 Sample Description 
                 (Kjeldahl) 
               
               
                   
               
             
             
               
                 405984 
                 
                   Bos taurus 
                 
                 Milk Protein Conc 80% 
                 80.37% 
               
               
                 410058 
                 (Cow) 
                 Milk Protein Conc 80% 
                 80.95% 
               
               
                 410057 
                   
                 Milk Protein Conc 80% 
                 79.89% 
               
               
                 409386 
                 
                   Pisum sativum 
                 
                 Pea Protein 80% 
                 85.85% 
               
               
                 404987 
                 (Pea) 
                 GRN Vegotein 80% 
                 79.25% 
               
               
                 407133 
                   
                 GRN Vegotein 80% 
                 80.17% 
               
               
                 408894 
                 
                   Oryza sativa 
                 
                 Rice Protein 80% 
                 83.71% 
               
               
                 409338 
                 (Rice) 
                 GRN Organic Oryzatein 90% 
                 85.25% 
               
               
                 405915 
                   
                 GRN Organic Oryzatein 90% 
                 85.84% 
               
               
                 405663 
                 
                   Glycine max 
                 
                 SoyPura 310 
                 92.10% 
               
               
                 409547 
                 (Soy) 
                 Supro 661 IP 
                 90.93% 
               
               
                 395810 
                   
                 Supro 661 IP 
                 91.18% 
               
               
                 410640 
                 None. Blend of 
                 iBCAA 2:1:1 
                    (0%) 
               
               
                 410641 
                 amino acids (LEU, 
                 iBCAA 2:1:1 
                    (0%) 
               
               
                 410642 
                 ILE, VAL). 
                 iBCAA 2:1:1 
                    (0%) 
               
               
                 409016 
                 None. 
                 WBT C4 Mass BR 
                    (0%) 
               
               
                 409101 
                   
                 WBT C4 Mass FP 
                    (0%) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Optimized MRM parameters used for marker peptide detection. 
               
             
          
           
               
                   
                   
                   
                 Q1 
                 Q3 
                 CE 
               
               
                 Protein (source) 
                 Peptide 
                 Amino Acid Sequence 
                 (m/z) 
                 (m/z) 
                 (V) 
               
               
                   
               
             
          
           
               
                 α-S1-Casein 
                 1 
                 FFVAPFPEVFKG 
                 692.9 
                 920.5 
                 29 
               
               
                 (Cow Milk) 
                   
                 (SEQ ID NO: 6) 
                   
                   
                   
               
               
                   
                 2 
                 YLGYLEQLLR 
                 634.4 
                 991.6 
                 33 
               
               
                   
                   
                 (SEQ ID NO: 7) 
                   
                   
                   
               
               
                   
                 3 
                 HQGLPQEVLNENLLR 
                 880.5 
                 1324.7 
                 51 
               
               
                   
                   
                 (SEQ ID NO: 8) 
                   
                   
                   
               
               
                   
               
               
                 β-Lactoglobulin 
                 1 
                 VYVEELKPTPEGDLEILLQK 
                 1157.1 
                 1453 
                 60 
               
               
                 (Cow Milk) 
                   
                 (SEQ ID NO: 9) 
                   
                   
                   
               
               
                   
                 2 
                 VLVLDTDYK 
                 533.3 
                 853.5 
                 23 
               
               
                   
                   
                 (SEQ ID NO: 10) 
                   
                   
                   
               
               
                   
                 3 
                 LIVTQTMK 
                 467.3 
                 707.4 
                 21 
               
               
                   
                   
                 (SEQ ID NO: 11) 
                   
                   
                   
               
               
                   
               
               
                 Vicilin 
                 1 
                 EGSLLLPHYNSR 
                 693.4 
                 773.6 
                 40 
               
               
                 (Pea) 
                   
                 (SEQ ID NO: 12) 
                   
                   
                   
               
               
                   
                 2 
                 GDFELVGQR 
                 510.8 
                 572.5 
                 26 
               
               
                   
                   
                 (SEQ ID NO: 13) 
                   
                   
                   
               
               
                   
                 3 
                 GPIYSNEFGK 
                 556.3 
                 844.5 
                 29 
               
               
                   
                   
                 (SEQ ID NO: 14) 
                   
                   
                   
               
               
                   
               
               
                 Glutelin 
                 1 
                 ALPNDVLANAYR 
                 658.9 
                 566.8 
                 26 
               
               
                 (Rice) 
                   
                 (SEQ ID NO: 15) 
                   
                   
                   
               
               
                   
                 2 
                 LQAFEPIR 
                 487.3 
                 732.5 
                 23 
               
               
                   
                   
                 (SEQ ID NO: 16) 
                   
                   
                   
               
               
                   
                 3 
                 GDEFGAFTPIQYK 
                 736.9 
                 1024.8 
                 33 
               
               
                   
                   
                 (SEQ ID NO: 17) 
                   
                   
                   
               
               
                   
               
               
                 Glycinin G1 
                 1 
                 VLIVPQNFVVAAR 
                 713.4 
                 1001.6 
                 33 
               
               
                 (Soy) 
                   
                 (SEQ ID NO: 19) 
                   
                   
                   
               
               
                   
                 2 
                 VFDGELQEGR 
                 575.3 
                 903.4 
                 29 
               
               
                   
                   
                 (SEQ ID NO: 19) 
                   
                   
                   
               
               
                   
                 3 
                 LNALKPDNR 
                 520.8 
                 629.3 
                 32 
               
               
                   
                   
                 (SEQ ID NO: 20) 
                   
                   
                   
               
               
                   
               
             
          
         
       
     
         [0051]    The chromatograms for negative control matrices were examined to assess the selectivity of the method. For each negative control matrix, the criteria was that no peaks for the marker peptides at the respective retention time could exceed 20% of the lowest qualifying MRL peak area. During MRL testing, it was determined that for specific peptides in RM-2 and FG-1 MRL values could not be assigned. The MRL peak areas for RM-1 spikes were used to assess the selectivity of RM-1 and RM-2 negative control samples while FG-2 spikes were used for FG-1 and FG-2 negative control samples. The selectivity results are shown in Table 11, with the percentage of the MRL peak area for any relevant peaks. Full tabulated results of selectivity are shown in Tables 12 and 13. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Summary of selectivity testing of negative control matrices. The results are 
               
               
                 expressed as percentage of the lowest qualifying MRL peak area in the 
               
               
                 appropriate matrix. For RM-1 and RM-2 negative control samples, the MRL 
               
               
                 peak areas in RM-1 were used. For FG-1 and FG-2 negative control samples, 
               
               
                 the MRL peak areas in FG-2 were used. For marker peptides that were not 
               
               
                 present in the negative control samples a value of 0% was assigned. 
               
             
          
           
               
                   
                 Cow Milk - 
                 Cow Milk - 
                   
                   
                   
               
               
                   
                 Casein 
                 Whey 
                 Rice 
                 Pea 
                 Soy 
               
             
          
           
               
                   
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
                   
               
             
          
           
               
                 RM-1 
                 9% 
                 7% 
                 14% 
                 0% 
                 0% 
                 7% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
               
               
                 RM-2 
                 10% 
                 8% 
                 19% 
                 3% 
                 19% 
                 9% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
               
               
                 FG-1 
                 11% 
                 5% 
                 12% 
                 0% 
                 11% 
                 0% 
                 0% 
                 0% 
                 0% 
                 13% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
               
               
                 FG-2 
                 10% 
                 7% 
                 9% 
                 0% 
                 10% 
                 10% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
                 0% 
               
               
                   
               
             
          
         
       
     
         [0052]    No peaks were observed in the negative control samples whose peak area was greater than 20% of the lowest qualifying MRL peak area. The method was therefore determined to be selective and free from reagent impurities or matrix effects. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 MRL Peak areas for spikes into negative control matrix 
               
             
          
           
               
                   
                 Cow Milk - 
                 Cow Milk- 
                   
                   
                   
               
               
                   
                 Casein 
                 Whey 
                 Rice 
                 Pea 
                 Soy 
               
             
          
           
               
                   
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
                   
               
             
          
           
               
                 RM-1 
                 116000 
                 67800 
                 5690 
                 1100 
                 1830 
                 2760 
                 509 
                 584 
                 97 
                 552 
                 471 
                 114 
                 698 
                 601 
                 81 
               
               
                 FG-2 
                 107000 
                 68100 
                 6200 
                 552 
                 1990 
                 2460 
                 81 
                 855 
                 49 
                 244 
                 244 
                 503 
                 1340 
                 341 
                 65 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 Peak areas for peaks detected in negative control matrix samples 
               
             
          
           
               
                   
                 Cow Milk - Casein 
                 Cow Milk - Whey 
                 Rice 
                 Pea 
                 Soy 
               
             
          
           
               
                   
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
                   
               
             
          
           
               
                 RM-1 
                 10300 
                 5040 
                 812 
                 0 
                 0 
                 195 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 RM-2 
                 11900 
                 5290 
                 1100 
                 33 
                 341 
                 244 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 FG-1 
                 11700 
                 3610 
                 717 
                 0 
                 211 
                 0 
                 0 
                 0 
                 0 
                 33 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 FG-2 
                 11200 
                 4430 
                 541 
                 0 
                 195 
                 244 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
               
             
          
         
       
     
       Example 4: Specificity 
       [0053]    The specificity of the qualitative method was established through examination of individual raw material samples from various protein sources. The criteria for specificity was that for each raw material sample, peaks for all three source marker peptides must be present with peak area greater than the MRL peak area, and that for other marker peptides no peaks with area greater than the MRL should be present. For each raw material sample, the signal was compared to the MRL peak areas in RM-1 spikes. Specificity results are shown in Table 14. Full specificity data sets are contained within Tables 15 and 16. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Specificity testing. Shown are the results for three lots of protein powder 
               
               
                 from each of the four source species. A “+” result was assigned 
               
               
                 to marker peptides whose peak areas was greater than the MRL. 
               
             
          
           
               
                   
                 Cow Milk - 
                 Cow Milk - 
                   
                   
                   
               
               
                   
                 Casein 
                 Whey 
                 Rice 
                 Pea 
                 Soy 
               
             
          
           
               
                   
                 SID 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
                   
               
             
          
           
               
                 Milk 
                 405984 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                   
                   
                   
                 + 
                   
                   
                   
                   
                   
               
               
                   
                 410058 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                   
                   
                   
                 + 
               
               
                   
                 410057 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 Rice 
                 408894 
                   
                   
                   
                   
                   
                   
                 + 
                 + 
                 + 
               
               
                   
                 409338 
                   
                   
                   
                   
                   
                   
                 + 
                 + 
                 + 
               
               
                   
                 405915 
                   
                   
                   
                   
                   
                   
                 + 
                 + 
                 + 
                   
                   
                 + 
               
               
                 Pea 
                 409386 
                   
                   
                   
                   
                 + 
                   
                 + 
                   
                   
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 404987 
                   
                   
                   
                   
                 + 
                   
                 + 
                   
                   
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 407133 
                   
                   
                   
                   
                 + 
                   
                 + 
                   
                   
                 + 
                 + 
                 + 
               
               
                 Soy 
                 405663 
                   
                   
                   
                   
                 + 
                   
                   
                   
                   
                   
                   
                   
                 + 
                 + 
                 + 
               
               
                   
                 409547 
                   
                   
                   
                   
                 + 
                   
                   
                   
                   
                   
                   
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 395810 
                   
                   
                   
                   
                 + 
                   
                   
                   
                   
                   
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 15 
               
               
                   
               
               
                 Specificity Testing (peak area) included are MRL peak areas for RM-1 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Cow Milk - Casein 
                 Cow Milk - Whey 
                 Rice 
               
             
          
           
               
                 Sample 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
               
                 BCAA:400640 
                 116000 
                 67800 
                 5690 
                 1100 
                 1830 
                 2760 
                 509 
                 584 
                 97 
               
               
                 405984_Milk 
                 20400000 
                 15500000 
                 5550000 
                 752000 
                 1830000 
                 1630000 
                 0 
                 0 
                 0 
               
               
                 410058_Milk 
                 21500000 
                 15600000 
                 5640000 
                 762000 
                 1850000 
                 1810000 
                 0 
                 0 
                 0 
               
               
                 410057_Milk 
                 21400000 
                 15700000 
                 5740000 
                 792000 
                 2040000 
                 1700000 
                 0 
                 0 
                 0 
               
               
                 408894_Rice 
                 8500 
                 5310 
                 1050 
                 0 
                 273 
                 476 
                 815000 
                 3050000 
                 86200 
               
               
                 409338_Rice 
                 8240 
                 5570 
                 652 
                 0 
                 365 
                 1000 
                 1020000 
                 6310000 
                 113000 
               
               
                 405915_Rice 
                 9480 
                 4710 
                 682 
                 130 
                 476 
                 963 
                 893000 
                 6340000 
                 107000 
               
               
                 409386_Pea 
                 10300 
                 6830 
                 1040 
                 179 
                 3110 
                 476 
                 1180 
                 0 
                 0 
               
               
                 404987_Pea 
                 8860 
                 6140 
                 931 
                 195 
                 3470 
                 446 
                 2220 
                 0 
                 0 
               
               
                 407133_Pea 
                 9160 
                 5680 
                 766 
                 0 
                 3600 
                 379 
                 1170 
                 195 
                 0 
               
               
                 405663_Soy 
                 14600 
                 6910 
                 1330 
                 260 
                 2800 
                 222 
                 352 
                 576 
                 0 
               
               
                 409547_Soy 
                 9070 
                 5070 
                 1230 
                 244 
                 1990 
                 769 
                 406 
                 303 
                 0 
               
               
                 395810_Soy 
                 9620 
                 4600 
                 678 
                 0 
                 2700 
                 555 
                 0 
                 401 
                 0 
               
               
                   
               
             
          
           
               
                   
                 Pea 
                 Soy 
               
             
          
           
               
                   
                 Sample 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
                   
               
               
                   
                 BCAA:400640 
                 552 
                 471 
                 114 
                 698 
                 601 
                 81 
               
               
                   
                 405984_Milk 
                 782 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 410058_Milk 
                 601 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 410057_Milk 
                 471 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 408894_Rice 
                 0 
                 309 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 409338_Rice 
                 357 
                 0 
                 0 
                 357 
                 0 
                 0 
               
               
                   
                 405915_Rice 
                 390 
                 0 
                 571 
                 0 
                 0 
                 0 
               
               
                   
                 409386_Pea 
                 1980000 
                 2460000 
                 559000 
                 4220 
                 1960 
                 555 
               
               
                   
                 404987_Pea 
                 2040000 
                 2290000 
                 527000 
                 1420 
                 731 
                 0 
               
               
                   
                 407133_Pea 
                 2170000 
                 2390000 
                 586000 
                 0 
                 0 
                 0 
               
               
                   
                 405663_Soy 
                 0 
                 441 
                 0 
                 5380000 
                 2400000 
                 656000 
               
               
                   
                 409547_Soy 
                 0 
                 0 
                 384 
                 8410000 
                 3120000 
                 969000 
               
               
                   
                 395810_Soy 
                 0 
                 1090 
                 238 
                 8610000 
                 3270000 
                 954000 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                 Specificity Results Expressed as Ratio of MRL peak area 
               
             
          
           
               
                   
                 Cow Milk - Casein 
                 Cow Milk - Whey 
                 Rice 
                 Pea 
                 Soy 
               
             
          
           
               
                 Sample 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
             
          
           
               
                 405984_Milk 
                 176 
                 229 
                 975 
                 684 
                 1000 
                 591 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 410058_Milk 
                 185 
                 230 
                 991 
                 693 
                 1011 
                 656 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 410057_Milk 
                 184 
                 232 
                 1009 
                 720 
                 1115 
                 616 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 408894_Rice 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1601 
                 5223 
                 885 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
               
               
                 409338_Rice 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2004 
                 10805 
                 1160 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 405915_Rice 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1754 
                 10856 
                 1099 
                 1 
                 0 
                 5 
                 0 
                 0 
                 0 
               
               
                 409386_Pea 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 2 
                 0 
                 0 
                 3587 
                 5223 
                 4904 
                 6 
                 3 
                 7 
               
               
                 404987_Pea 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 4 
                 0 
                 0 
                 3696 
                 4862 
                 4623 
                 2 
                 1 
                 0 
               
               
                 407133_Pea 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 2 
                 0 
                 0 
                 3931 
                 5074 
                 5140 
                 0 
                 0 
                 0 
               
               
                 405663_Soy 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 7708 
                 3993 
                 8079 
               
               
                 409547_Soy 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 3 
                 12049 
                 5191 
                 11933 
               
               
                 395810_Soy 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 2 
                 2 
                 12335 
                 5441 
                 11749 
               
               
                   
               
             
          
         
       
     
         [0054]    For 3 out of the 12 samples, no marker peptides other than the source peptides were seen above the respective MRLs. For these samples, the method behaved as expected. For 8 out of the 12 samples tested, one or two peptides from a set of non-source marker peptides were seen above the MRL. While the method did not pass the specificity criteria for these samples, identification relies upon peaks at greater than MRL level for all three of the specific marker peptides for that protein source. In those cases where only one (or even two) of the three maker peptide for a specified source is seen, the lack of signal for the other marker peptide(s) indicates that the specific protein is unlikely present in the sample. Thus, specificity of identification is maintained even while the single marker peptide specificity did not perform as expected. 
         [0055]    In one case (SID 409386, pea) all three peptides were observed for both pea and soy above the MRL. In this pea sample, the signal for all three pea peptides was &gt;3,500 times the MRL, while for the soy peptides the signal was only 3-7 times the MRL. These soy levels suggest the possibility that the sample contained low level contaminants. The explanation for this observation is not that there is not method specificity, but in fact may be due to the sample itself. This sample is not a true standard which further supports this conclusion, but does not confirm it. 
       Example 5: Precision 
       [0056]    The precision of the qualitative method was evaluated by examining the % RSD for marker peptide peak areas for intraday analysis of both high and low protein level samples. On one day, six replicate preparations of the initial protein mix (high) and the 1000 ppm spiked composite RM-1 (low) were taken through digestion. The next day these samples were analyzed in parallel. The summarized results are shown in Table 17. Full precision data sets are contained within Table 18. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 17 
               
             
             
               
                   
               
               
                 Precision testing. Shown is the relative standard deviation from intraday 
               
               
                 testing of high (pro mix) and low (RM-1, 1000) protein samples 
               
             
          
           
               
                 Sample 
                 Cow Milk-Casein 
                 Cow Milk-Whey 
                 Rice 
                 Pea 
                 Soy 
               
             
          
           
               
                 Name 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
               
                 Low 
                 9% 
                 15% 
                 15% 
                 29% 
                 12% 
                 16% 
                 28% 
                 19% 
                 179% 
                 15% 
                 9% 
                 3% 
                 22% 
                 19% 
                 66% 
               
               
                 RM-1, 1000 
               
               
                 % RSD 
               
               
                 High 
                 4% 
                  5% 
                  5% 
                  6% 
                  8% 
                 10% 
                  4% 
                  5% 
                  6% 
                  5% 
                 7% 
                 7% 
                  5% 
                  5% 
                  6% 
               
               
                 PRO MIX 
               
               
                 % RSD 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 18 
               
               
                   
               
               
                 Precision Testing, Tabulated Data of Peak Areas 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Milk-Casein 
                 Milk-Whey 
                 Rice 
               
             
          
           
               
                 Sample Name 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
               
                 RM-1, 1000, #1 
                 118000 
                 80600 
                 10200 
                 909 
                 3280 
                 5390 
                 790 
                 996 
                 0 
               
               
                 RM-1, 1000, #2 
                 118000 
                 56000 
                 10200 
                 714 
                 2810 
                 4680 
                 336 
                 1210 
                 0 
               
               
                 RM-1, 1000, #3 
                 96600 
                 54400 
                 6840 
                 1080 
                 3390 
                 4900 
                 525 
                 747 
                 0 
               
               
                 RM-1, 1000, #4 
                 101000 
                 61300 
                 9090 
                 587 
                 2710 
                 3700 
                 527 
                 823 
                 179 
               
               
                 RM-1, 1000, #5 
                 109000 
                 66300 
                 10800 
                 1340 
                 2790 
                 3600 
                 471 
                 763 
                 0 
               
               
                 RM-1, 1000, #6 
                 121000 
                 63400 
                 10200 
                 860 
                 3600 
                 4490 
                 598 
                 990 
                 65 
               
               
                 AVG 
                 110600 
                 63667 
                 9555 
                 915 
                 3097 
                 4460 
                 541 
                 922 
                 41 
               
               
                 SD 
                 10084 
                 9423 
                 1441 
                 268 
                 374 
                 696 
                 150 
                 178 
                 73 
               
               
                 RSD 
                 9% 
                 15% 
                 15% 
                 29% 
                 12% 
                 16% 
                 28% 
                 19% 
                 179% 
               
               
                 PRO MIX, #1 
                 13900000 
                 8300000 
                 2120000 
                 279000 
                 592000 
                 803000 
                 143000 
                 211000 
                 13800 
               
               
                 PRO MIX, #2 
                 15400000 
                 9340000 
                 2360000 
                 317000 
                 718000 
                 990000 
                 155000 
                 244000 
                 15700 
               
               
                 PRO MIX, #3 
                 14900000 
                 9130000 
                 2260000 
                 318000 
                 709000 
                 1010000 
                 148000 
                 238000 
                 14400 
               
               
                 PRO MIX, #4 
                 15200000 
                 9020000 
                 2410000 
                 303000 
                 732000 
                 1000000 
                 158000 
                 247000 
                 15400 
               
               
                 PRO MIX, #5 
                 15700000 
                 9560000 
                 2460000 
                 332000 
                 763000 
                 1100000 
                 159000 
                 237000 
                 13900 
               
               
                 PRO MIX, #6 
                 14700000 
                 8920000 
                 2370000 
                 308000 
                 707000 
                 995000 
                 154000 
                 240000 
                 15700 
               
               
                 AVG 
                 14966667 
                 9045000 
                 2330000 
                 309500 
                 703500 
                 983000 
                 152833 
                 236167 
                 14817 
               
               
                 SD 
                 631401 
                 431451 
                 122311 
                 17942 
                 58374 
                 97263 
                 6178 
                 12891 
                 889 
               
               
                 RSD 
                 4% 
                  5% 
                  5% 
                  6% 
                  8% 
                 10% 
                  4% 
                  5% 
                  6% 
               
               
                   
               
             
          
           
               
                   
                 Pea 
                 Soy 
               
             
          
           
               
                   
                 Sample Name 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
                   
               
               
                   
                 RM-1, 1000, #1 
                 3410 
                 4240 
                 1090 
                 1340 
                 4830 
                 49 
               
               
                   
                 RM-1, 1000, #2 
                 4630 
                 4560 
                 1190 
                 817 
                 4180 
                 1090 
               
               
                   
                 RM-1, 1000, #3 
                 3260 
                 3900 
                 1130 
                 1190 
                 4030 
                 585 
               
               
                   
                 RM-1, 1000, #4 
                 3850 
                 4190 
                 1140 
                 1060 
                 3730 
                 771 
               
               
                   
                 RM-1, 1000, #5 
                 4470 
                 4590 
                 1110 
                 1590 
                 3690 
                 254 
               
               
                   
                 RM-1, 1000, #6 
                 3450 
                 5010 
                 1140 
                 1330 
                 5930 
                 616 
               
               
                   
                 AVG 
                 3845 
                 4415 
                 1133 
                 1221 
                 4398 
                 561 
               
               
                   
                 SD 
                 582 
                 388 
                 34 
                 265 
                 856 
                 370 
               
               
                   
                 RSD 
                 15% 
                 9% 
                 3% 
                 22% 
                 19% 
                 66% 
               
               
                   
                 PRO MIX, #1 
                 688000 
                 808000 
                 194000 
                 2410000 
                 980000 
                 227000 
               
               
                   
                 PRO MIX, #2 
                 787000 
                 919000 
                 214000 
                 2820000 
                 1110000 
                 252000 
               
               
                   
                 PRO MIX, #3 
                 771000 
                 920000 
                 225000 
                 2720000 
                 1070000 
                 255000 
               
               
                   
                 PRO MIX, #4 
                 764000 
                 951000 
                 228000 
                 2640000 
                 1080000 
                 253000 
               
               
                   
                 PRO MIX, #5 
                 780000 
                 986000 
                 236000 
                 2660000 
                 1130000 
                 264000 
               
               
                   
                 PRO MIX, #6 
                 775000 
                 980000 
                 225000 
                 2650000 
                 1090000 
                 271000 
               
               
                   
                 AVG 
                 760833 
                 927333 
                 220333 
                 2650000 
                 1076667 
                 253667 
               
               
                   
                 SD 
                 36526 
                 65022 
                 14706 
                 135351 
                 52026 
                 14989 
               
               
                   
                 RSD 
                  5% 
                 7% 
                 7% 
                  5% 
                  5% 
                  6% 
               
               
                   
                   
               
             
          
         
       
     
         [0057]    For the composite sample spiked at 1000 ppm, the majority of marker peptides, 10 of 15, showed acceptable precision % RSD≦20%. The % RSD value represents the combined variability of the sample preparation, instrument performance and sample homogeneity. These results suggest that precision is only as reliable as the results in the table for the 5 peptides that exceeded acceptable precision. 
         [0058]    Typically samples are expected to contain 20-80% protein, so the protein mix sample is appropriate for examining the precision of the method for regular analysis. All marker peptides in the protein mix sample had % RSD≦10%. This indicates that at the higher protein levels (˜20% protein), the qualitative identification method performed with acceptable precision. 
       Example 6: Reinjection Reproducibility 
       [0059]    To assess reinjection reproducibility, the primary extracts for the raw material samples that had been stored in the refrigerator at 4° C. for five days were taken through the final dilution step and analyzed. The results for the stored sample were to be deemed acceptable if peaks for all three marker peptides were present with peak areas greater than the MRL. 
         [0060]    In each reinjected sample, the appropriate marker peptides had peak areas greater than the MRL. The peak areas were compared to the original data and showed relative responses of approximately 70-130%. These results indicate that for qualitative identification the extracts can be reexamined up to 5 days later if stored appropriately. The reinjection results are shown in Table 19. Full reinjection data sets are contained within Tables 20 and 21. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 19 
               
             
             
               
                   
               
               
                 Reinjection reproducibility testing. Shown are the results for the source marker peptide 
               
               
                 for the reinjection of raw material samples. Results are expressed as a percentage 
               
               
                 of the original marker peptide response. 
               
             
          
           
               
                   
                 Cow Milk-Casein 
                 Cow Milk-Whey 
                 Rice 
                 Pea 
                 Soy 
               
             
          
           
               
                   
                 SID 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
             
          
           
               
                 Milk 
                 405984 
                 108 
                 109 
                 116 
                 127 
                 119 
                 72 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 410057 
                 107 
                 110 
                 117 
                 122 
                 124 
                 72 
               
               
                   
                 410058 
                 104 
                 106 
                 113 
                 120 
                 112 
                 78 
               
               
                 Rice 
                 405915 
                   
                   
                   
                   
                   
                   
                 115 
                 117 
                 114 
               
               
                   
                 408894 
                   
                   
                   
                   
                   
                   
                 116 
                 117 
                 115 
               
               
                   
                 409338 
                   
                   
                   
                   
                   
                   
                 119 
                 114 
                 107 
               
               
                 Pea 
                 404987 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 117 
                 120 
                 114 
               
               
                   
                 407133 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 105 
                 117 
                 111 
               
               
                   
                 409386 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 103 
                 108 
                 97 
               
               
                 Soy 
                 395810 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 115 
                 108 
                 96 
               
               
                   
                 405663 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 111 
                 106 
                 97 
               
               
                   
                 409547 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 116 
                 109 
                 100 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 20 
               
               
                   
               
               
                 Original Injections of raw material samples (peak area) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Cow Milk-Casein 
                 Cow Milk-Whey 
                 Rice 
               
             
          
           
               
                 Sample 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
               
                 BCAA:400640 
                 116000 
                 67800 
                 5690 
                 1100 
                 1830 
                 2760 
                 509 
                 584 
                 97 
               
               
                 405984_Milk 
                 20400000 
                 15500000 
                 5550000 
                 752000 
                 1830000 
                 1630000 
                 0 
                 0 
                 0 
               
               
                 410058_Milk 
                 21500000 
                 15600000 
                 5640000 
                 762000 
                 1850000 
                 1810000 
                 0 
                 0 
                 0 
               
               
                 410057_Milk 
                 21400000 
                 15700000 
                 5740000 
                 792000 
                 2040000 
                 1700000 
                 0 
                 0 
                 0 
               
               
                 408894_Rice 
                 8500 
                 5310 
                 1050 
                 0 
                 273 
                 476 
                 815000 
                 3050000 
                 86200 
               
               
                 409338_Rice 
                 8240 
                 5570 
                 652 
                 0 
                 365 
                 1000 
                 1020000 
                 6310000 
                 113000 
               
               
                 405915_Rice 
                 9480 
                 4710 
                 682 
                 130 
                 476 
                 963 
                 893000 
                 6340000 
                 107000 
               
               
                 409386_Pea 
                 10300 
                 6830 
                 1040 
                 179 
                 3110 
                 476 
                 1180 
                 0 
                 0 
               
               
                 404987_Pea 
                 8860 
                 6140 
                 931 
                 195 
                 3470 
                 446 
                 2220 
                 0 
                 0 
               
               
                 407133_Pea 
                 9160 
                 5680 
                 766 
                 0 
                 3600 
                 379 
                 1170 
                 195 
                 0 
               
               
                 405663_Soy 
                 14600 
                 6910 
                 1330 
                 260 
                 2800 
                 222 
                 352 
                 576 
                 0 
               
               
                 409547_Soy 
                 9070 
                 5070 
                 1230 
                 244 
                 1990 
                 769 
                 406 
                 303 
                 0 
               
               
                 395810_Soy 
                 9620 
                 4600 
                 678 
                 0 
                 2700 
                 555 
                 0 
                 401 
                 0 
               
               
                   
               
             
          
           
               
                   
                 Pea 
                 Soy 
               
             
          
           
               
                   
                 Sample 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
                   
               
               
                   
                 BCAA:400640 
                 552 
                 471 
                 114 
                 698 
                 601 
                 81 
               
               
                   
                 405984_Milk 
                 782 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 410058_Milk 
                 601 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 410057_Milk 
                 471 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 408894_Rice 
                 0 
                 309 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 409338_Rice 
                 357 
                 0 
                 0 
                 357 
                 0 
                 0 
               
               
                   
                 405915_Rice 
                 390 
                 0 
                 571 
                 0 
                 0 
                 0 
               
               
                   
                 409386_Pea 
                 1980000 
                 2460000 
                 559000 
                 4220 
                 1960 
                 555 
               
               
                   
                 404987_Pea 
                 2040000 
                 2290000 
                 527000 
                 1420 
                 731 
                 0 
               
               
                   
                 407133_Pea 
                 2170000 
                 2390000 
                 586000 
                 0 
                 0 
                 0 
               
               
                   
                 405663_Soy 
                 0 
                 441 
                 0 
                 5380000 
                 2400000 
                 656000 
               
               
                   
                 409547_Soy 
                 0 
                 0 
                 384 
                 8410000 
                 3120000 
                 969000 
               
               
                   
                 395810_Soy 
                 0 
                 1090 
                 238 
                 8610000 
                 3270000 
                 954000 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 21 
               
               
                   
               
               
                 Reinjection of raw material samples (peak area) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Milk-Casein 
                 Milk-Whey 
                 Rice 
               
             
          
           
               
                 Sample 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
               
               
                 405984_Milk_RI 
                 22100000 
                 16900000 
                 6440000 
                 957000 
                 2180000 
                 1180000 
                 0 
                 0 
                 0 
               
               
                 410057_Milk_RI 
                 22900000 
                 17100000 
                 6580000 
                 931000 
                 2290000 
                 1310000 
                 0 
                 0 
                 0 
               
               
                 410058_Milk_RI 
                 22300000 
                 16600000 
                 6490000 
                 948000 
                 2290000 
                 1320000 
                 0 
                 0 
                 0 
               
               
                 405915_Rice_RI 
                 5350 
                 3200 
                 731 
                 0 
                 0 
                 1050 
                 936000 
                 3570000 
                 98500 
               
               
                 408894_Rice_RI 
                 5620 
                 2860 
                 698 
                 0 
                 393 
                 1390 
                 1180000 
                 7400000 
                 130000 
               
               
                 409338_Rice_RI 
                 6670 
                 3700 
                 536 
                 0 
                 0 
                 1230 
                 1060000 
                 7210000 
                 115000 
               
               
                 404987_Pea_RI 
                 7570 
                 4880 
                 877 
                 0 
                 3300 
                 0 
                 0 
                 0 
                 0 
               
               
                 407133_Pea_RI 
                 6210 
                 3780 
                 698 
                 0 
                 3390 
                 854 
                 0 
                 0 
                 0 
               
               
                 409386_Pea_RI 
                 6630 
                 3100 
                 422 
                 0 
                 4060 
                 514 
                 0 
                 211 
                 0 
               
               
                 395810_Soy_RI 
                 13200 
                 5900 
                 2050 
                 0 
                 3070 
                 610 
                 0 
                 0 
                 0 
               
               
                 405663_Soy_RI 
                 8030 
                 3210 
                 1050 
                 0 
                 3690 
                 292 
                 0 
                 0 
                 0 
               
               
                 409547_Soy_RI 
                 7270 
                 3330 
                 1250 
                 0 
                 3100 
                 0 
                 0 
                 0 
                 0 
               
               
                   
               
             
          
           
               
                   
                 Pea 
                 Soy 
               
             
          
           
               
                   
                 Sample 
                 Pep1 
                 Pep2 
                 Pep3 
                 Pep1 
                 Pep2 
                 Pep3 
               
               
                   
                   
               
               
                   
                 405984_Milk_RI 
                 444 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 410057_Milk_RI 
                 703 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 410058_Milk_RI 
                 706 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 405915_Rice_RI 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 408894_Rice_RI 
                 385 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 409338_Rice_RI 
                 568 
                 0 
                 544 
                 0 
                 0 
                 0 
               
               
                   
                 404987_Pea_RI 
                 2310000 
                 2950000 
                 635000 
                 4430 
                 1530 
                 0 
               
               
                   
                 407133_Pea_RI 
                 2140000 
                 2670000 
                 583000 
                 2220 
                 341 
                 0 
               
               
                   
                 409386_Pea_RI 
                 2230000 
                 2590000 
                 568000 
                 0 
                 0 
                 0 
               
               
                   
                 395810_Soy_RI 
                 0 
                 0 
                 0 
                 6170000 
                 2580000 
                 630000 
               
               
                   
                 405663_Soy_RI 
                 0 
                 1390 
                 0 
                 9310000 
                 3320000 
                 941000 
               
               
                   
                 409547_Soy_RI 
                 0 
                 0 
                 0 
                 10000000 
                 3550000 
                 957000 
               
               
                   
                   
               
             
          
         
       
     
         [0061]    All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. 
         [0062]    While described in certain embodiments, the present disclosure can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the methods described herein using its general principles. Further, this disclosure is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and that fall within the limits of the appended claims and their legal equivalents. 
       BIBLIOGRAPHIC REFERENCES 
       [0000]    
       
         1 Haug A, Høstmark A T, Harstad O M, A; Høstmark, A T; Harstad, O M (25 Sep. 2007). “Bovine milk in human nutrition—a review”. Lipids Health Dis 6: 25. doi:10.1186/1476-511X-6-25. 
         2 McWilliam H., Valentin F., Goujon M., Li W., Narayanasamy M., Martin J., Miyar T. and Lopez R. (2009) Web services at the European Bioinformatics Institute. Nucleic Acids Research 37: W6-W10. 
       
     
         [0065]    (α-S1-Casein, http://www.ebi.ac.uk/interpro/entry/IPR026999) 
         [0066]    (β-Lactoglobulin, http://www.ebi.ac.uk/interpro/entry/IPR002447)
   3 Peter R Shewry and Rod Case eds., (1999). Seed Proteins. Kluwer Academic Publishers, Netherlands.   4 O&#39;Kane F E1, Happe R P, Vereijken J M, Gruppen H, van Boekel M A., (2004). Characterization of pea vicilin. 1. Denoting convicilin as the alpha-subunit of the  Pisum vicilin  family. J Agric Food Chem. 2004 May 19; 52(10):3141-8.   5 Jiang C, Cheng Z, Zhang C, Yu T, Zhong Q, Shen J Q, Huang X, (2014). Proteomic analysis of seed storage proteins in wild rice species of the  Oryza  genus. Proteome Sci. 2014 Nov. 30; 12(1):51. doi: 10.1186/s12953-014-0051-4.   6 Wolf, W. J. (1972) Purification and properties of the proteins. In: Soybeans: Chemistry and Technology. Vol. 1. Smith, A. K. and Circle, S. J., eds. Avi Publishing Co. Inc., Westport, Conn.   7 Baker, P. R. and Clauser, K. R. http://prospector.ucsf.edu.   8 Madden T.  The BLAST Sequence Analysis Tool.  2002 Oct. 9 [Updated 2003 Aug. 13]. In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (Md.): National Center for Biotechnology Information (US); 2002—. Chapter 16. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21097/   9 Protein authenticity, http://www.authentechnologies.com/applications/species-identification/