Abstract:
Disclosed is a method for the separation, identification and quantification of multiple proteins in pancreatin active pharmaceutical ingredient samples.

Description:
This patent application claims the benefit of provisional patent application No. 61/528,582 filed on Aug. 29, 2011. 
    
    
     FIELD OF THE INVENTION 
     Background of the Invention 
     Pancreatin is a mixture of several digestive enzymes produced by the exocrine cells of the pancreas. It is composed of amylase, lipase and protease. The pancreas gland produces both endocrine secretions that enter the blood stream and exocrine secretions that enter the duodenum. Whereas the endocrine secretions comprise hormones such as insulin and glucagon, the exocrine secretions to a large part are made up of enzymes necessary for digestion of food in the duodenum. 
     Without these enzymes (normally produced by the human pancreas), a substantial portion of undigested food simply passes through the digestive tract and provides no nutritional benefit. Pancreatin can be manufactured from the pancreas of either a pig or a cow. Porcine pancreatin juice is closest to that of humans, with high proportions of lipase and alpha-amylase in comparison with other mammals. Therefore, porcine pancreatin is made only from the pancreas of pigs, and is used to treat conditions in which pancreatic secretions are deficient, such as surgical pancreatectomy, pancreatitis and cystic fibrosis. Pancreatin has been claimed to help with food allergies, celiac disease, autoimmune disease, cancer and weight loss. Pancreatin is sometimes called “pancreatic acid”, although it is neither a single chemical nor an acid. 
     Pancreatin enzyme products (PEPs) of porcine or bovine origin have been available in the United States for the treatment of exocrine pancreatic insufficiency (EPI) since before the enactment of the Federal Food, Drug, and Cosmetic Act of 1938 (the Act). With the exception of one PEP approved in 1996, PEPs have been marketed without New Drug Applications (NDAs) and were considered as dietary supplements. In recent years, however, the use of PEPs has been severely restricted in the US and Europe due to being derived from animal product, with a risk of viral transmission and bioactivity poorly characterized and standardized. The Food and Drug Administration (FDA) considers that an Over The Counter (OTC) monograph is not sufficient to adequately regulate these drug products and to standardize enzyme bioactivity, safety and effectiveness. The FDA&#39;s guidance for the industry requires all pharmaceutical companies marketing pancreatic enzymes for pancreatin deficiency to be approved under New Drug Applications. Since April 2010, PEPs are available by prescription only and only PEPs approved by the FDA remain on the market. 
     To be approved, an NDA must meet the requirements in 21 CFR §314.50 regarding chemistry, manufacturing and controls (CMC) information. The drug substance should be adequately characterized using chemical, physical and biological testing. Batch-to-batch consistency with respect to chemical identity, biological activity of different classes of enzymes including specific activity, and identity and purity levels should be demonstrated. Since Pancreatin is referenced in a new NDA, the agency expects the pancreatin Drug Master File (DMF) to meet current ICH Q6B requirements for specifications. Specifications for the drug substance should include tests for identity, biological activity of different classes of enzyme, purity and other relevant attributes. Because of the complexity of pancreatin extract product, it is unlikely that currently-available physiological and biological analytical tools would be able to demonstrate that the active ingredients in pancreatic extract products from two different batches/manufacturers are the same. Current United States Pharmacopeia (USP) monograph tests are insufficient to characterize the API to meet the ICH guideline. The new regulatory guidelines now require better methods to characterize pancreatin API. 
     SUMMARY OF THE INVENTION 
     In light of the foregoing, it is an object of the present invention to provide a method for the separation of proteins in a sample of pancreatin active pharmaceutical ingredient (“API”), the method comprising reverse phase-high performance liquid chromatography (“RP-HPLC”). In particular, proteins including trypsin, chymotrypsin, elastase, carboxypeptidase-B, phospholipase-A2, lipase, colipase, and amylase can be separated and identified using the methods of the invention. Separation of pancreatin proteins according to the methods can be achieved on, for example, a C 4  RP-HPLC column. 
     It is yet another object of the invention to provide a method for the separation and identification of proteins in a sample of pancreatin API, the method comprising separating proteins from a pancreatin API sample via RP-HPLC and identifying each protein by one or more methods of co-chromatography (spiking), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption ionization-time of flight mass spectroscopy (MALDI-TOF-TOF MS) and Western Blotting. In accordance with the method, proteins including trypsin, chymotrypsin, elastase, carboxypeptidase-B, phospholipase-A2, lipase, colipase, and amylase can be separated and identified using the methods of the invention. Also in accordance with the method, separation of the pancreatin proteins can be achieved on, for example, a C 4  RP-HPLC column. 
     It is still another object of the invention to provide a method for the analysis of proteins in a sample of pancreatin API, the method comprising separating proteins from a pancreatin API sample via RP-HPLC; identifying each protein by one or more methods of co-chromatography (spiking), SDS-PAGE, MALDI-TOF-TOF MS and Western Blotting; and calculating amounts of each identified protein present in the sample of pancreatin. In accordance with the method, proteins including trypsin, chymotrypsin, elastase, carboxypeptidase-B, phospholipase-A2, lipase, colipase, and amylase can be separated and identified using the methods of the invention. Also in accordance with the method, separation of the pancreatin proteins can be achieved on, for example, a C 4  RP-HPLC column. Further in accordance with the method, calculation of amounts of each identified protein can be achieved using various software, such as, for example, Empower software. 
     Accordingly, it will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the following objects can be viewed in the alternative with respect to any one aspect of this invention. 
     Other objects, features, benefits and advantages of the present invention will be apparent from this summary and the following descriptions of certain embodiments, and will be readily apparent to those skilled in the art. Such objects, features, benefits and advantages will be apparent from the above as taken into conjunction with the accompanying examples, data, and all reasonable inferences to be drawn therefrom. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
         FIG. 1  is the RP-HPLC analysis of pancreatin API sample 1. 
         FIG. 2  is a protein profile of pancreatin API a) sample 1 and 2) sample 2. 
         FIG. 3  is a table with relative amounts of various proteins in RP-HPLC peaks of pancreatin API sample 1. 
         FIG. 4  depicts the molecular weights of RP-HPLC pancreatin API protein peak fractions (4-12% SDS-PAGE). M1: SeeBlue plus protein marker; M2: MultiMark; A/S: Appliend sample for HPLC; V: Void; 1-25: HPLC pancreatin API Fraction #1-25. 
         FIG. 5  is a table with the molecular weights of pancreatin API proteins collected from RP-HPLC; *Relative abundance (%) of a protein band in a peak; “1208”=pancreatin API. 
         FIG. 6  is the RP-HPLC analysis of trypsin standard. 
         FIG. 7  depicts standard curves generated using Sigma trypsin. 
         FIG. 8  is an SDS-PAGE analysis of purified porcine pancreatic a) lipase; b) colipase; and c) carboxypeptidase-B, wherein lane 1 represents unpurified enzyme and lanes 2-4 represent purified enzymes. 
         FIG. 9  is RP-HPLC analysis of spiked pancreatic API spiked with commercially-available standards of procine enzymes (spiking in red): a) PLA 2 ; b) trypsin; c) elastase; and d) amylase. 
         FIG. 10  is a table that identifies individual proteins by MS and Western Blotting detected by primary and secondary antibodies. 
         FIG. 11  is a Western Blot analysis of pancreatin peaks. 
         FIG. 12  is the result of the development of an RP-HPLC method: a) Göke; b) developed method; and c) modified Göke. 
         FIG. 13  depicts the optimum running conditions for SDS-PAGE. 4-12% Bis-Tris NuPAGE of colipase and carboxypeptidase B; running conditions: reduced, 1×MES running buffer and 150 V for 55 minutes. 
         FIG. 14  depicts SDS-PAGE analysis of HPLC-separated fractions for the determination of molecular weight of pancreatin API proteins (4-12% SDS-PAGE of C 4  RP-HPLC fractions); SB: SeeBlue plus protein standard; MM: MultiMark plus protein standard; AS: HPLC applied sample: sample 1. 
         FIG. 15  is a 4-12% Bis-Tris NuPAGE showing solubilized and unsolubilized proteins from sample 1. Lanes—1: SeeBlue Plus protein Marker; 2 &amp; 3: 20 μL soluble proteins of 1 mg/mL and 5 mg/mL solution, respectively; 4 &amp; 5: 200 μL of unsolublized proteins of 1 mg/mL and 100 μL of 5 mg/mL solution, respectively; 6: Standard Bovine Chymotrypsin. 
         FIG. 16  shows the Western Blot of anti-PLA 2 . Lanes—1: MagicMark; 2 and 7: RP-HPLC loading sample 1; 3: Porcine PLA 2  from Sigma; 4: Pancreatin API peaks 4 and 5; 5: Pancreatin API peak 3; 6: HPLC-pancreatin API peak 1 and 2; 8: MultiMark. 
         FIG. 17  shows the Western Blot of anti-elastase. Lanes—1: SeeBlue Plus2 protein marker; 2: RP-HPLC loading sample 1; 3: Porcine Elastase from Sigma; 4: Pancreatin API peak fraction 14; 5: Pancreatin API peak fraction 15; 6: Pancreatin API peak fraction 16; 7: Pancreatin API peak fraction 17; 8: Pancreatin API peak fraction 18; 9: RP-HPLC loading sample 1; 10: MagicMark. 
         FIG. 18  shows the Western Blot of anti-lipase. S: solubilized Pancreatin API proteins; uns: unsolubilized Pancreatin API proteins; P3, P10-14: HPLC peak fractions; Std1: porcine lipase from Elastin Products Company; Std2: porcine lipase from USP; Std3: porcine lipase from Boehringer Mannhein; Pancreatin API: HPLC loading sample 1. Arrows indicate the full length porcine lipase: 49.9 kDa. 
         FIG. 19  shows the Western Blot of anti-CP-B. SB: SeeBlue Plus 2 protein marker; AS: HPLC-Applied sample 1; P 13-P23: HPLC peak fractions; MM: MagicMark; Std: Porcine Carboxypeptidase B from Sigma. 
         FIG. 20  shows the Western Blot of anti-amylase. Lanes—1: SeeBlue Plus2 Protein Marker; 2: HPLC loading sample 1; 3: Porcine α-Amylase from Sigma; 4: HPLC-1208 peak fraction 19. Arrow indicates the porcine amylase (full length). 
         FIG. 21  shows the Western Blot of anti-trypsin. Lanes—1: SeeBlue plus2 protein Marker; 2: Standard porcine trypsin; 3: HPLC-pancreatin API peaks 6 &amp; 7. 
         FIG. 22  Western Blot of pancreatin API-1 and -2 (API peaks 1 and 2). 
         FIG. 23  Western Blot of pancreatin API-3. 
         FIG. 24  Western Blot of analysis of pancreatin API-4 and 5. 
         FIG. 25  Western Blot of analysis of pancreatin API-6 and -7. 
         FIG. 26  Western Blot of pancreatin API-8 and -9. 
         FIG. 27  Western Blot of pancreatin API-10. 
         FIG. 28  Western Blot of pancreatin API-12. 
         FIG. 29  a) Western Blot and MS b) of pancreatin API-13. 
         FIG. 30  Western Blot of pancreatin API-14. 
         FIG. 31  Western Blot of pancreatin API-15. 
         FIG. 32  Western Blot of pancreatin API-17. 
         FIG. 33  Western Blot of pancreatin API-19. 
         FIG. 34  Western Blot of pancreatin API-21. 
         FIG. 35  Western Blot of pancreatin API-22. 
         FIG. 36  Western Blot of pancreatin API-24. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Reverse Phase-High Performance Liquid Chromatography 
     Accordingly, the present invention includes a RP-HPLC method to separate proteins in a pancreatin API sample. The method comprises treating a pancreatin sample with a solvent to afford a soluble protein sample; fractionating the soluble protein sample using reverse phase-high performance liquid chromatography (RP-HPLC); collecting each of the fractionated proteins individually; and characterizing each of the fractionated proteins using one or methods selected from the group consisting of co-chromatography, SDS-PAGE, Western Blotting and MALDI-TOF-MS. The RP-HPLC mode separates proteins based on their hydrophobic characteristics. Proteins bind to RP-HPLC columns in aqueous mobile phase and are eluted from the column by increasing the hydrophobicity of mobile phase. The stationary phase (resin) is made up of hydrophobic alkyl chains (—CH 2 —CH 2 —CH 2 —CH 3 ). There are three common chain lengths: C 4 , C 8 , and C 18 , but others exist. Proteins are large molecules and can have multiple hydrophobic moieties to interact with the column. The shorter chain length (C 4 ), therefore, is often more appropriate for protein separation. 
     Illustrating certain non-limiting aspects and embodiments of the invention, a C 4  column (Vydac 214MS54, 250×4.6 mm, 5 micron, or water BEH C4, 250×4.6 mm, 3.5 micron) is used for the separation of pancreatin API proteins. Pancreatin proteins are separated by running a linear-segmented gradient of acetonitrile containing 0.1% trifluoroacetic acid (TFA). The eluted proteins are detected at 280 nm. The relative amounts of individual peaks are quantified using Waters Empower software. Quantification is based on integrated peak area of protein peaks using a threshold set at 0.5-120, and peak width set at 20-100. 
     Generally, to identify the protein peaks in pancreatin API, known protein standard solutions are spiked into the pancreatin sample and chromatography procedures are performed under the same elution conditions. 
     Sample Preparation for RP-HPLC 
     Pancreatin API samples are prepared as described below in Example 1. The soluble fraction of pancreatin API after centrifugation is used for RP-HPLC analysis. Soluble and insoluble fractions of pancreatin API are analyzed by SDS-PAGE to examine the components in both fractions (see Example 2). The results show that more than 95% of pancreatin API proteins are solubilized in the method procedure, and the majority components of the insoluble proteins are likely truncated lipase and chymotrypsin-like proteases as identified by SDS-PAGE or Western blot. 
     To optimize separation of pancreatin proteins on RP-HPLC, different columns, C 4 , C 8  and C 18 , and various elution gradients are tested. The best resolution is obtained from a C 4  column using elution conditions described in Table 1. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 (Column: C 4 ; Temperature: 40° C.; Sample: 1-2 mg/mL in 0.1% 
               
               
                 TFA; Autosampler temperature: 10° C.; 
               
               
                 Mobile phase: 
               
               
                 Solvent A: 0.1% TFA in PureLab water 
               
               
                 Solvent B: 0.1% TFA in Acetonitrile) 
               
               
                 Elution Conditions: 
               
             
          
           
               
                 Time (min) 
                 Flow (mL/min) 
                 Solvent A (%) 
                 Solvent B (%) 
               
               
                   
               
             
          
           
               
                 0 
                 1.0 
                 85 
                 15 
               
               
                 14 
                 1.0 
                 65 
                 35 
               
               
                 28 
                 1.0 
                 60 
                 40 
               
               
                 37 
                 1.0 
                 50 
                 50 
               
               
                 37.5 
                 1.2 
                 10 
                 91 
               
               
                 38.0 
                 1.2 
                 10 
                 90 
               
               
                 39.0 
                 1.0 
                 85 
                 15 
               
               
                 50.0 
                 1.0 
                 85 
                 15 
               
               
                   
               
             
          
         
       
     
     The RP-HPLC analysis shows the major twenty five protein peaks present in pancreatin API sample 1 ( FIG. 1 ; Injection: 100 μL of supernatant of pancreatin API; Column: C 4  (4.6×250 mm); Solvent A: 0.1% TFA in PureLab water; Solvent B: 0.1% TFA in acetonitrile; Elution method: method above detection: 280 nm). Similar HPLC profiles are obtained from two different batches (sample 1; SPL-1208-1257)(sample 2; SPL-1208-1211) of pancreatin API ( FIG. 2 ; Loading: 100 μL of 1 mg/mL sample 1; Elution: developed method). The relative amounts of each protein can be calculated using an integration threshold of 10 to 120 and peak width of 25 to 100. The settings can be adjusted as needed until an appropriate baseline position of the peak is obtained.  FIG. 3  lists the results calculated from three replicates using threshold of 10 and a peak width of 100 (Injection: 100 μL of 2 mg/mL sample 1; Calculation: Peak width=100, threshold=10; *Relative amount (%)=Individual peak area/total peak area×100). The maximum % RSD is less than 7% for all 25 peaks, indicating that the method is reproducible with precision. 
     Determination of Molecular Weights (MW) 
     Electrophoretic separations by SDS-PAGE are performed on peaks collected from RP-HPLC to determine the MW of the proteins. Sodium dodecyl sulfate (SDS) is an anionic detergent which denatures proteins and binds to proteins specifically in a mass ratio of 1.4:1. Therefore, the number of SDS molecules that bind to a protein is proportional to the number of amino acids of the protein. A reducing agent (Dithiothreitol, or “DTT”) in the sample loading buffer cleaves disulfide bonds covalently linking other protein subunits. Under reducing and denaturing conditions, all proteins in the sample are resolved according to size (MW) during SDS-PAGE. 
     A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured protein and its R f . The R f  is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front. Determination of molecular weight (Mr) is achieved by plotting a standard curve of distance migrated vs. log 10  MW for protein standards, and read off the log Mr of the protein to be identified after measuring distance migrated on the same gel. Quantification of proteins is based on integrated optical density of a band. The Kodak 1D 3.6 software is designed based on this principle and is used for this data analysis. 
     Different SDS-polyacrylamide gels and different running buffers are tested for separation. The following combinations of gel and running buffer are evaluated: 4-20% Tris-glycine gel with Tris-glycine buffer; 10% Bis-Tris NuPAGE with MOPS buffer; 12% Bis-Tris NuPAGE with MOPS buffer; 4-12% Bis-Tris NuPAGE with MOPS buffer; and 4-12% Bis-Tris NuPAGE with MES buffer. The best separation for pancreatin API proteins is obtained with 4-12% Bis-Tris NuPAGE using MES buffer (see Example 4 below). 
     Determination of molecular weight (MW) is done by SDS-PAGE. The molecular weights of the HPLC-pancreatin API peak fractions (the fractions numbers match the elution profile in  FIG. 2 ) are presented in  FIG. 4  and  FIG. 5  (see also Example 4). Void fraction collected from the largest peak in pancreatin API-HPLC chromatograms contains only small fragments (less than 3 kDa) with relatively small quantities. 
     Western Blotting 
     Western blot analysis is used to characterize pancreatin proteins. Because of the ability of antibody molecules to recognize and bind to antigen very specifically, this antibody-binding specificity can be used to identify proteins. Proteins across different species with similar functions may share sequence homology. Therefore, antibodies against the protein in one species could also react with the protein in other species with similar function. 
     In a typical Western blot analysis, pancreatin API proteins are separated on a denaturing SDS polyacrylamide gel and are transferred (blotted) to a nitrocellulose membrane. The membrane is then exposed sequentially to solutions containing primary antibody, followed by a secondary antibody coupled to an enzyme. The membrane is then soaked in a substrate solution to develop a colored reaction, which results in identifying the antigen as a band. 
     Experiments are carried out using a Novagen HIS-Tag AP Western Kit. Primary antibodies used in this study are listed in Table 2. Immunoreactive proteins are detected using Alkaline Phosphatase (AP)-conjugated goat anti-mouse or anti-rabbit immunoglobulin (Promega, Madison, Wis.). All antibodies are diluted to the appropriate level in blocking solution. 
                                     TABLE 2                           Vender,           Antibody   Source   Immunogen   Cat. #   Dilution                   Anti-PLA 2     Rabbit   Porcine   Upstate   1:500               pancreatic   Cat. #06-                   phospholipase   150                   A 2                 Anti-   Rabbit   Porcine   Biogenesis   1:4000       Carboxypeptidase       pancreatic   Cat. #1810-           B       Carboxypeptidase   0130                   B               Anti-Trypsin   Rabbit   Human pancreatic   Fitzgerald   1:1000               Trypsin   Cat. #70-                       XR77           Anti-   Rabbit   Human   Fitzgerald   1:1000       Chrymotrypsin       pancreatic   Cat. #20-                   Chymotrypsin   CR79           Anti-Amylase   Rabbit   Human   Fitzgerald   1:3000               pancreatic   Cat. #20-                   amylase   AR07           Anti-Lipase   Rabbit   Human   Fitzgerald   1:100               pancreatic   Cat. #10-                   lipase   L50           Anti-Elastase   Rabbit   Human   Fitzgerald   1:500               pancreatic   Cat. #20-                       ER66                    
MALDI-TOF-TOF Mass Spectrometry
 
     Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF MS) techniques are utilized to identify proteins in pancreatin API. MALDI-TOF mass spectrometry is a relatively novel tool for protein characterization. The technique is based upon an ultraviolet absorbing matrix. The matrix and biomolecules (peptide fragments generated using amino-acid specific proteolytic enzyme, for example) are mixed at a molecular level in an appropriate solvent with a ˜104-fold molar excess of the matrix. The solvent prevents aggregation of the polymer. The sample/matrix mixture is placed onto a sample probe tip. The solvent is then removed, leaving co-crystallized biomolecules homogeneously dispersed within matrix molecules. When the pulsed laser beam is tuned to the appropriate frequency, the energy is transferred to the matrix causing desorption and ionization of the biomolecules. The ionized biomolecules are accelerated in an electric field and enter the flight tube where they are then separated in time and reach the detector. An analyzer measures the time-of-flight (TOF). The flight time of an ion is related to its mass-to-charge ratio. In this way, each molecule yields a distinct signal; thus, mass spectra can be generated. The masses of the peptides are obtained from mass spectrometry. Proteins are identified by matching the observed sequences of the peptides to NCBI proteins database (3,794,285 sequences in the database, see Example 5). The pancreatin API protein samples are prepared and sent out for MS analysis at the Mass Spectrometry Facility (Biotechnology Center, University of Wisconsin-Madison). 
     Quantitation of Trypsin in Pancreatin API 
     Due to the availability of high-purity commercial standards, the trypsin in pancreatin API samples can be quantitated using an external standard. 
     To select a good external standard, porcine trypsin is purchased from Sigma and US Biological (USB). The standards are dissolved in HPLC solvent A and analyzed by RP-HPLC. Trypsin from Sigma has a higher purity than that of USB ( FIG. 6 ). The Sigma product (Cat. # T0303) contains 3 major peaks of trypsin (retention times 17 to 18 min). The chromatograms also contain a broad peak at 41.5 minutes due to gradient wash and re-equilibration. The USB trypsin (Cat. #22715) contains two major peaks of trypsin (retention time between 17 and 18 min) and several other large peaks. Because of its higher purity, Sigma trypsin is used for quantification of trypsin in SPL API samples. 
     Standard curves are generated using Sigma trypsin. Serial dilutions of trypsin standards, 0.0625, 0.125, 0.25, 0.5, 0.75 1.0 and 2.0 mg/mL are prepared and analyzed by RP-HPLC. Two injections are performed for each concentration and are averaged. Data is calculated for linearity between peak area and trypsin amount (each of the three peaks and their sums). The linear range of the standard curve for trypsin is 0.156 to 5.0 μg. In this range, R 2  values of 0.9982 to 0.9989 are obtained (Table 3 and  FIG. 7 ). 
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 RP-HPLC Analysis of Trypsin Standard 
               
             
          
           
               
                 Trypsin 
                 Peak Area 
               
             
          
           
               
                 (μg) 
                 Peak 1  
                 Peak 2 
                 Peak 3 
                 Sum 
               
               
                   
               
             
          
           
               
                 0.3125 
                 808 
                 2488 
                 13855 
                 17151 
               
               
                 0.3125 
                 846 
                 2630 
                 14736 
                 18212 
               
               
                 0.625 
                 1765 
                 5438 
                 29741 
                 36944 
               
               
                 0.625 
                 1741 
                 5144 
                 29598  
                 36483 
               
               
                 1.25 
                 3739 
                 11147 
                 61024 
                 75910 
               
               
                 1.25 
                 3674 
                 11360 
                 60570 
                 75604 
               
               
                 1.875 
                 6080 
                 17615 
                 91933 
                 115628 
               
               
                 1.875 
                 6010 
                 16723 
                 91808 
                 114541 
               
               
                 2.5 
                 7514 
                 22891 
                 120092  
                 150497 
               
               
                 2.5 
                 7877 
                 23140  
                 121756 
                 152773 
               
               
                 5 
                 14925 
                 44476 
                 233429 
                 292830 
               
               
                 5 
                 15040  
                 45932 
                 232793  
                 293765 
               
               
                   
               
             
          
         
       
     
     Using the standard curve, the amounts of trypsin are quantified in pancreatin API, sample 1 and sample 2. As seen in Table 4, similar levels of trypsin are detected in these two different lots of SPL API-1208. The % RSD values of trypsin concentration from three injections of 1208-1257 ranged from 0.4 to 11.9% for three separated trypsin peaks. The % RSD value was much lower (0.5%) when all three peak areas were summed. 
                                                                     TABLE 4                   Trypsin   Peak Area            (μg)    Peak 1   Peak 2   Peak 3   Sum                    0.3125   808   2488   13855   17151       0.3125    846   2630   14736    18212       0.625   1765   5438   29741    36944       0.625   1741   5144   29598    36483       1.25   3739   11147   61024   75910       1.25   3674   11360    60570   75604       1.875   6080   17615   91933   115628       1.875   6010   16723    91808   114541       2.5   7514   22891   120092   150497       2.5   7877   23140   121756   152773       5   14925   44476   233429   292830       5   15040    45932   232793   293765                    
Identification of Individual Proteins
 
     The RP-HPLC peak fractions of pancreatin API sample 1 are manually collected and lyophilized. The dried protein fractions are dissolved in 1× lithium dodecylsulfate (LDS) sample buffer containing 50 mM dithiothreitol (DTT). Proteins are identified using the methods of co-chromatography (spiking), SDS-PAGE, Western Blotting and MALDI-TOF-MS. 
     Co-Chromatography (Spiking Studies) 
     Porcine pancreatic enzymes, trypsin, elastase, carboxypeptidase-B (CPB), carboxypeptidase-A (CPA), phospholipase-A2 (PLA 2 ), lipase, colipase, and amylase are commercially available. The purities of trypsin, elastase, PLA 2  and amylase are high enough for spiking; the others are further purified. 
     Purification of Standard Pancreatic Proteins: Lipase, Colipase and Carboxypeptidase-B by SDS-PAGE 
     Porcine pancreatic enzymes, lipase (USP, Cat. #1494079, Lot # I1E327), colipase (Boehringer Mannhein) and carboxypeptidase-B (Sigma, Cat. # C9584, Lot #108H74061) are purchased. After separation on SDS-PAGE under reducing conditions, proteins are revealed by SimplyBlue SafeStain (Invitrogen, Cat. # LC6060). Protein bands of interest are excised and eluted with 100 mM sodium acetate (NaOAc) containing 0.1% SDS and 10 mM DTT, pH 7.8. Eluted proteins are dialyzed overnight at 4° C. against 100 mM NaOAc buffer, pH 7.8 to remove SDS. The SDS-PAGE analysis ( FIG. 8 ) of the purified pancreatic enzymes shows a purity of greater than 95% for all three enzymes. 
     The pancreatin API samples are spiked with commercially-available standards of porcine enzymes: trypsin, elastase, PLA 2  and amylase. The results show that PLA 2  co-eluted with RP-HPLC peaks 1&amp;2, trypsin co-eluted with peaks 4-7, elastase co-eluted with peak 15 and amylase co-eluted with peak 19 (see  FIG. 9 ). 
     The purified lipase, colipase and CPB are also analyzed by RP-HPLC. When spiking the purified enzymes into pancreatin API proteins, the results are inconclusive because a significant degradation of pancreatin API proteins occurs at pH 6.0 (the higher pH is the result of mixing purified enzyme and pancreatin API protein sample). 
     Protein Identification by Western Blotting 
     Western Blotting or immunoblotting procedures combine the resolution of gel electrophoresis with the specificity of antibody detection. An antibody locks on to a specific protein even when surrounded by others proteins. This method is used to determine the presence and the molecular weights of proteins in pancreatin API samples. 
     The primary and secondary antibodies used in this study are listed in  FIG. 10 . The RP-HPLC separated pancreatin API proteins that react with these antibodies are summarized in  FIG. 10 . (See Example 5). 
     Protein Identification by MALDI-TOF-TOF Mass Spectrometry 
     Matrix Assisted Laser Desorption Ionization Time-of-flight-Time-of-flight (MALDI-TOF-TOF) Mass Spectrometry (MS) is used to determine both the accurate peptide mass and peptide sequences of pancreatin API proteins. 
     Trypsin, chymotrypsin, elastase (three types: elastase, elastase-I and elastase-II), carboxypeptidase (two types: carboxypeptidase-A and carboxypeptidase-B), phospholipase-A 2 , lipase, colipase, and amylase are identified in a pancreatin API sample ( FIG. 10 ). In addition, porcine pancreatic lithostathine (an inhibitor of calcium carbonate crystals and bacterial aggregation in the pancreas) is also identified (see Example 5). 
     The MS results generated from peak 13 did not match any sequences in the database, but immunoblot analysis showed that it reacted specifically with human anti-chymotrypsin antibody ( FIG. 11 ). This suggests that this protein is a member of the chymotrypsin family, like elastase, trypsin and kallikrein. No known porcine chymotrypsin peptide sequences are found in sequence databases. 
     In summary, all proteins revealed in the RP-HPLC elution profile of pancreatin API are identified by methods of co-chromatography (HPLC spiking), Western blotting, or MS. No unexpected proteins are detected. Some proteins existed in different sizes (full length or truncated) and isoforms. 
     EXAMPLES 
     Example 1 
     RP-HPLC 
     Separation and identification of panreatin API proteins. Quantification of Trypsin in pancreatin APIs using an external Trypsin standard. 
     Materials and Equipment: 
     1) Porcine Trypsin: Sigma, Cat. # T0303; 
     2) Pancreatin API samples 
     3) Trifluoroacetic acid (TFA): Pierce, Cat. #28904; 
     4) PureLab water; 
     5) Acetonitrile: J. T. Baker, Cat. #9829, or equivalent; 
     6) 50 mL Sterile centrifuge tubes: Fisher, Cat. #06-443-18; 
     7) 15 mL Sterile centrifuge tubes: Corning, Cat. #430766; 
     8) 1.5 mL Eppendorf tubes; 
     9) C 4  column: Vydac, Cat. #214MS54, 250×4.6 mm, 5 micron; 
     10) Waters Alliance HPLC 2695 System with 2487 UV detector; 
     11) Eppendorf centrifuge: CENT-7000005 
     Mobile Phase: 
     Solvent A (0.1% TFA in H 2 O): add 1 mL TFA to 1 L PureLab water; 
     Solvent B (0.1% TFA in acetonitrile): add 1 mL TFA to 1 L acetonitrile. 
     Preparation of Protein Samples: 
     Pancreatin API protein samples: 1 mg/mL. Weigh 30 mg of pancreatin API powder is put into a 50-mL centrifuge tube. 30 mL of Solvent A is subsequently added to the tube, and proteins are extracted by shaking on a rocker shaker (AI-7000008, set speed at 4) at room temperature for 30 minutes. An aliquot of the solution (1 mL) is transferred into a microcentrifuge tube and centrifuged at 13,000 rpm for 10 minutes. The supernatant (800 μL) is carefully removed and used for analysis. 
     Trypsin Standard Solutions. 10±0.1 mg of Sigma Trypsin is dissolved in 5 mL of Solvent A. The resulting solution is centrifuged at 13,000 rpm in an Eppendorf centrifuge for 5 minutes, resulting in a standard solution of 2 mg/mL. Table 5 shows the standard dilutions of Trypsin prepared using this stock solution (2.5 μL of each standard injected for RP-HPLC analysis). 
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                   
                 Volume of 
                 Volume and 
                 Final Trypsin 
               
               
                   
                 Solvent A 
                 source of 
                 Concentration 
               
               
                 Tube # 
                 (mL) 
                 Trypsin (mL) 
                 (μg/mL) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 0 
                 2 mL of Std 
                 2000 
               
               
                   
                   
                 (2 mg/mL) 
                   
               
               
                 2 
                 0 
                 1.0 mL of 
                 1500 
               
               
                   
                   
                 tube 1 + l mL 
                   
               
               
                   
                   
                 of tube 3 
                   
               
               
                 3 
                 1.0 
                 1.0 mL of 
                 1000 
               
               
                   
                   
                 tube 1 
                   
               
               
                   
                   
                 dilution 
                   
               
               
                 4 
                 0 
                 1.0 mL of 
                 750 
               
               
                   
                   
                 tube 3 + 1 mL 
                   
               
               
                   
                   
                 of tube 5 
                   
               
               
                 5 
                 1.0 
                 1.0 mL of 
                 500 
               
               
                   
                   
                 tube 3 
                   
               
               
                   
                   
                 dilution 
                   
               
               
                 6 
                 1.0 
                 1.0 mL of 
                 250 
               
               
                   
                   
                 tube 4 
                   
               
               
                   
                   
                 dilution 
                   
               
               
                 7 
                 1.0 
                 1.0 mL of 
                 125 
               
               
                   
                   
                 tube 5 
                   
               
               
                   
                   
                 dilution 
                   
               
               
                 8 
                 1.0 
                 1.0 mL of 
                 62.5 
               
               
                   
                   
                 tube 6 
                   
               
               
                   
                   
                 dilution 
               
               
                   
               
             
          
         
       
     
     RP-HPLC Procedure: 
     The RP-HPLC method is equilibrated and samples are used with the method (See Table 1 above). 
     Trypsin peak area of the two major peaks are integrated from each standard run using appropriate settings of threshold and peak width (adjust settings until appropriate baseline positions of the peaks are obtained). Sum the area of two peaks. 
     The standard curves of the sum of peak area vs. Trypsin amount (μg) are plotted. Printed best-fit equations and values of R 2  generated.
 
 Y=mX+b;  
 
     Y: Peak area; X: amount of Trypsin (μg). 
     After the amount of Trypsin, X, is measured, the average Trypsin peak area of replicates is measured. The Trypsin amount is then determined using the equation of standard curves. Calculation of Trypsin Concentration: mg/g dry weight.
 
Trypsin= Y−b/m× 10*
 
*Conversion factor=1/100 μL (injection)×1000 μL (sample)
 
     Example 2 
     Development of RP-HPLC Method 
     Comparison of Elution Conditions for Pancreatin API Separation: 
     Methods: developed method (Table 1, above); Göke (Table 6); and modified Göke (Table 7). 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 Time  
                 Flow  
                 Solvent  
                 Solvent  
               
               
                   
                 (min) 
                 (ml/min) 
                 A (%) 
                 B (%) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 0 
                 1.0 
                 100 
                 0 
               
               
                   
                 3 
                 1.0 
                 67 
                 33 
               
               
                   
                 5 
                 1.0  
                 67  
                 33 
               
               
                   
                 17 
                 1.0 
                 61 
                 39 
               
               
                   
                 30 
                 1.0 
                 60  
                 40 
               
               
                   
                 35 
                 1.0 
                 58 
                 42 
               
               
                   
                 36 
                 1.0  
                 54 
                 46 
               
               
                   
                 42 
                 1.0 
                 52  
                 48 
               
               
                   
                 45 
                 1.0 
                 52 
                 48 
               
               
                   
                 50 
                 1.0 
                 45  
                 55 
               
               
                   
                 52 
                 1.0  
                 45  
                 55 
               
               
                   
                 60 
                 1.0 
                 27  
                 73 
               
               
                   
                 61 
                 1.0  
                 27  
                 73 
               
               
                   
                 62 
                 1.0  
                 100 
                 0 
               
               
                   
                 65 
                 1.0 
                 100 
                 0 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 Time  
                 Flow  
                 Solvent 
                 Solvent  
               
               
                   
                 (min) 
                 (ml/min) 
                 A (%) 
                 B (%) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 0 
                 1.0 
                 0 
                 100 
               
               
                   
                 3 
                 1.0 
                 85 
                 15 
               
               
                   
                 15 
                 1.0 
                 67 
                 33 
               
               
                   
                 17 
                 1.0 
                 61 
                 39 
               
               
                   
                 30 
                 1.0 
                 50 
                 50 
               
               
                   
                 35 
                 1.0 
                 50 
                 50 
               
               
                   
                 36 
                 1.0 
                 10 
                 90 
               
               
                   
                 39 
                 1.0 
                 10 
                 90 
               
               
                   
                 40 
                 1.0 
                 100 
                 0 
               
               
                   
                 50 
                 1.0 
                 100 
                 0 
               
               
                   
                   
               
             
          
         
       
     
     The results of the three methods are depicted in  FIG. 12 . The best separation is obtained using the developed method. 
     Example 3 
     SDS-PAGE Analysis 
     The SDS-PAGE procedures were used for: 1) analysis of proteins in 1208 samples; 2) purification of target proteins; 3) quantification of proteins; 4) determination of the molecular weight of RP-HPLC fractions; 
     5) determination of the purity of protein preparations; 6) Western Blotting; and 7) in-gel digestion of proteins for MS. 
     Materials: 
     Novex NuPAGE® 4-12% Bis-Tris mini-gels #NP0321 (Invitrogen.) 
     Novex NuPAGE 12% Bis-Tris #NP 0341 (Invitrogen) 
     Novex 4-12% Tris Glycine #EC60252 (Invitrogen) 
     Novex NuPAGE® antioxidant, 15 mL #NP0005 (Invitrogen) 
     Novex NuPAGE® MES running buffer (20×) #NP0002 (Invitrogen) 
     Novex NuPAGE® MOPS running buffer (20×) #NP0001 (Invitrogen) 
     Novex TrisGlycine running buffer (20×) #LC2675 (Invitrogen) 
     Novex NuPAGE® LDS sample buffer (4×) #NP0007 (Invitrogen) 
     Novex NuPAGE® Transfer buffer (20×) #NP0006 (Invitrogen 
     SeeBlue Plus MW standard #LC5925 (Invitrogen) 
     Novex Simple Blue Safe Stain #LC6060 (Invitrogen) 
     Dithiothreitol (DTT), 99%, ACS reagent #45,777-9 (Aldrich Chemical Co.) 
     Buffer Preparation: 
     a) MES SDS (1×) Running Buffer—40 mL of Novex NuPAGE® MES SDS buffer concentrate (20×) is diluted to 800 mL with PureLab water in a 1-liter graduated mixing cylinder. The cylinder is sealed with a stopper and slowly inverted five times to mix (minimize foaming). 
     B) MESS DS (1×) Running Buffer Plus Antioxidant—200 mL of MES SDS (1×) running buffer (see above) is transferred to a 250-mL Erlenmeyer flask. 0.5 mL of Novex NuPAGE® antioxidant is added and the flask is swirled gently to mix. 
     Sample Loading Buffer (2×): 
     Prepared fresh on the day of use. For 1 mL of loading buffer, the following components are combined in a microcentrifuge tube: 500 μL Novex NuPAGE® LDS (4×) sample buffer, 200 μL DTT solution (0.5 M), and 300 μL PureLab water. If a larger amount is required, volumes are scaled accordingly. 0.5 M DTT is prepared by dissolving 0.386 g (2.5 mmoles) dithiothreitol in 5.0 mL of PureLab water. 0.5-mL aliquots are stored frozen in microcentrifuge tubes for future use. 
     Sample Loading Buffer (1×): 
     For a 1 mL: 250 μL Novex NuPAGE® LDS (4×) sample buffer; 100 μL 0.5 M DTT solution; 650 μL PureLab water. 
     Sample Preparation: 
     A) Pancreatin API—Soluble and insoluble pancreatin API protein samples are prepared as follows: 50-60 mg of pancreatin API powder are weighed out and put into a 50-mL tube. 0.1% TFA in PureLab water (Mobile Phase A) is added to make a solution of 2 mg/mL. The tube is sealed and the sample is shaken on a rocker shaker (AI-7000008, speed at 4) at room temperature for 30 minutes. 1 mL of the solution is transferred into a centrifuge tube and centrifuged at 13,000 rpm for 10 minutes. 100 μL of supernatant is carefully removed to a fresh centrifuge tube and 100 μL of 2× sample loading buffer (sample defined as soluble proteins) is added. The rest of the supernatant is removed, 1.0 mL of 0.1% TFA is added, mixed, and centrifugation is repeated one more time. All supernatant is then remove and discarded. The pellet is resuspended with 1 mL of 1× sample loading buffer (sample defined as insoluble protein). All samples are heated at 70±2° C. for 10 minutes in a heating block. 
     B) Dried HPLC Fraction—The dried HPLC fraction is resuspended with 1× of sample loading buffer (20 μL/mL of collected volume prior to drying) and transferred to a fresh microcentrifuge tube. The tubes are sealed and heated at 70±2° C. for 10 minutes in a heating block. 
     Running SDS-PAGE: 
     After heating, samples are spin loaded at 13,000 rpm briefly (30 seconds) and 10 μL (1208 mix) or 20 μL (HPLC fractions or purified protein) is loaded on 4-12% Bis-Tris NuPAGE gel. The upper chamber is filled in Novex XCell SureLock® mini-cell with 200 mL of “SDS Running Buffer (1×) Plus Antioxidant,” and the lower chamber is filled with 600 mL of 1× running buffer. The gel(s) is run at 150 V for 1 hour. 
     The optimum running conditions for SDS-PAGE is depicted in  FIG. 13 . 4-12% Bis-Tris with MES buffer is chosen for separation of pancreatin API proteins. SDS-PAGE analysis of HPLC-separated fractions for the determination of molecular weight of pancreatin API proteins is depicted in  FIG. 14  (4-12% SDS-PAGE of C 4  RP-HPLC fractions). 
     To check if pancreatin proteins are well solubulized in 0.1% TFA for HPLC analysis, fractions of solubilized and insolubilized proteins are prepared as follows in Scheme A: 
     
       
                 
         
             
             
         
      
     
     4-12% Bis-Tris NuPAGE shows solubilized and unsolubilized proteins from sample 1 ( FIG. 15 ). Protein bands of Chymotrypsin are identified by analyzing the samples side-by-side with the standards. The Lipase band is identified by Western blotting. 
     Density analysis of the scanned SDS-PAGE image indicates that 97% of pancreatin proteins are solubilized when samples are prepared at 1 mg/mL. The amount of solubilized protein is slightly decreased to 90% in samples of 5 mg/mL. Therefore, preparation of samples at 1 mg/mL for HPLC analysis is preferred. 
     The fraction of unsolubilized protein contains mainly chymotrypsin and truncated lipases, which are also well represented in the fraction of solubilized protein, as detected by RP-HPLC. 
     Example 4 
     Western Blotting 
     Western Blotting is used to determine the presence of a specific protein in different samples with a specific primary antibody. The experiments are carried out using a Novex Western blot kit (Invitrogen, Cat. #70972). 
     Preparation of Transfer Buffer (1×): 
     50 mL 20× NuPAGE transfer buffer (Invitrogen, Cat. #nbNP0006); 100 mL Methanol; 849 mL pure H 2 O; 1 mL antioxidant. 
     Procedure for Western Blotting: 
     A) Preparation of sample for SDS-PAGE (loading samples)—5 μL 4×LDS sample buffer; 2 μL 0.5 M DTT; 13 μL Protein solution. Samples are heated at 70° C., 10 minutes; spin at 13,000 rpm, 1 minute. 
     B) Run SDS-PAGE (4-12% Bis-Tris NuPAGE)—10 μL of loading samples are loaded. The upper chamber is filled with 200 mL of 1×MES running Buffer and 0.5 mL antioxidant is added. The lower chamber is filled with 600 mL MES buffer and the gels are run at 150 V for 55-60 minutes. 
     C) Western Blotting—Nitrocellulose membrane filter paper sandwich (Invitrogen Cat. #LC 2000) and 4 blotting pads (Novex Cat. #NaEI9052) are soaked in 1× transfer buffer containing 10% methanol (prepared from 20× NuPAGE transfer buffer, Invitrogen, Cat. # NP0006-1) at room temperature for 5 minutes with shaking (on the rocker shaker, AI-7000008, set speed at 2). After SDS-PAGE, the gels are transferred to the membrane in 1× transfer buffer at room temperature, 15 V for 50 minutes. The membrane is blocked in blocking solution at room temperature for 2 hours (membrane is flipped over after 1 hour) or overnight (placed in refrigerator after 2 hours). The primary antibody is diluted with 1× blocking solution (15 mL): The membrane is incubated in diluted primary antibody for 2 hours at room temperature on a shaker (the membrane is flipped over after 1 hour). The membrane is then washed with 1×TBSTT (25 mL/membrane) for 5 minutes, 3 times, and then washed with 1×TBSTT, 20 mL of 1×TBS for 5 minutes. The membrane is then incubated in 15 mL of diluted secondary antibody (1:7,000 in blocking solution) for 1 hour at room temperature on a shaker (the membrane is flipped over after 30 minutes). The membrane is subsequently washed with 1×TBSTT, 20 mL of 1×TBS for 5 minutes. During the last wash, 15 mL of developing solution is prepared for each membrane as follows: 15 mL pure H 2 O; 60 μL NBT solution; 60 μL BCIP solution. The wash buffer is drained and the blot is placed, protein side up, in the developing solution. 
       FIG. 16  shows the blot of anti-PLA 2  (immunogen: Porcine PLA 2 ; working solution: 1:500 in block solution; antibody incubation: 2 hours at room temperature for primary antibody and 1 hour for secondary antibody (1:7500)). Anti-PLA 2  antibody recognizes the 14 kDa protein which is present in pancreatin API peaks 1 and 2. 
       FIG. 17  shows the blot of anti-elastase (immunogen: human pancreatic elastase; working solution: 1:1000 in block solution; antibody incubation: 2 hours at room temperature for primary antibody and 1 hour for secondary antibody (1:7500)). Anti-elastase antibody recognizes the 25.9 kDa protein which is present in pancreatin API peaks 14-18. 
       FIG. 18  shows the blot of anti-lipase (immunogen: human pancreatic lipase—full length; working solution: 1:100 in block solution; antibody incubation: 2 hours at room temperature for primary antibody and 1 hour for secondary antibody (1:7500)). Full-length porcine lipase (49.9 kDa) and truncated lipase are detected by anti-lipase antibody in pancreatin API mixture (solubilized and unsolubilized fractions), HPLC-peak fractions 3, 10-14. 
       FIG. 19  shows the blot of anti-CP-B (immunogen: human porcine carboxypeptidase B; working solution: 1:4000 in block solution; antibody incubation: 2 hours at room temperature for primary antibody and 1 hour for secondary antibody (1:7500)). Anti-CP-B antibody recognizes the 35 kDa protein which is present in pancreatin API peaks 21-23.  FIG. 20  shows the blot of anti-amylase (immunogen: human porcine amylase; working solution: 1:4000 in block solution; antibody incubation: 2 hours at room temperature for primary antibody and 1 hour for secondary antibody (1:7500)). Anti-amylase antibody recognizes the 51 kDa protein mix, porcine standard amylase and pancreatin API-peak 19 (reported porcine amylase 50-54 kDa). 
       FIG. 21  shows the blot of anti-trypsin (immunogen: human porcine trypsin; working solution: 1:1000 in block solution; antibody incubation: 2 hours at room temperature for primary antibody and 1 hour for secondary antibody (1:7500)). Proteins in pancreatin API peaks 6 and 7 reacted with human pancreatic anti-trypsin. 
     Example 5 
     MALDI-TOF-TOF MS 
     Preparation of pancreatin API samples for MALDI-TOF-TOF MS: 
     Samples—Sample 1 fractions are lyophilized and collected from C 4  RP-HPLC. 
     Sample preparation—20 μL of 1×LDS*/mL is collected, heated at 70° C. for 10 minutes, and centrifuged at 13,000 rpm for 1 minute. *1×LDS sample buffer with 50 mM DTT: for 1 mL 250 μL of 4×LDS sample buffer; 100 μL of 0.5 M DTT; 650 μL of pure water. 
     SDS-PAGE—4-12% Bis-Tris NuPAGE. 
     Running buffer—1×MES. 
     Running condition: 150 V constant, 1 hour under reducing conditions. 
     Staining—1 hour with Simple Blue. 
     Enzymatic Digestion and MS analysis: 
     “In Gel” digestion and mass spectrometric analysis is done at the Mass Spectrometry Facility [Biotechnology Center, University of Wisconsin-Madison]. The digestion is performed as outlined on the website http://www.biotech.wisc.edu/ServicesResearch/MassSpec/ingel.htm. 
     In short, Coomassie G-250 (Colloidal) stained gel pieces are de-stained completely in MeOH/H 2 O/NH 4 HCO 3  [50%:50%:100 mM], dehydrated for 10 min in ACN/H 2 O/NH 4 HCO 3  [50%:50%:25 mM] then once more for 1 minute in 100% ACN, dried in a Speed-Vac for 5 minutes, reduced in 25 mM DTT [Dithiotreitol in 25 mM NH 4 HCO 3 ] for 30 minutes at 56° C., alkylated with 55 mM IAA [Iodoacetamide in 25 mM NH 4 HCO 3 ] in darkness at room temperature for 30 minutes, washed twice in H 2 O for 1 minute, equilibrated in 25 mM NH 4 HCO 3  for 1 minute, dehydrated for 10 minutes in ACN/H 2 O/NH 4 HCO 3  [50%:50%:25 mM] then once more for 1 minute in 100% ACN, dried again and rehydrated with 20 μL of trypsin solution [20 ng/μL trypsin (Sequence Grade Modified from PROMEGA Corp.) in 25 mM NH 4 HCO 3 ]. The digestion is conducted overnight [18 hrs] at 37° C. and subsequently terminated by acidification with equal volume of 2.5% TFA [Trifluoroacetic Acid]. Peptides generated from digestion are extracted in two subsequent steps: first with equal volume of 0.1% TFA (˜50 μL) and vigorous vortexing for 15 minutes and then with the same volume of ACN/H 2 O/TFA [70%:25%:5%] and vortexing. The collected peptide solution is dried completely in a Speed-Vac then re-suspended in 50 μL of 0.1% TFA and solid-phase extracted (ZipTip® C18 pipette tips Millipore, Billerica, Mass.). Peptides are eluted off the C 18  zip tip with acetonitrile/H 2 O/TFA (70%:25%:0.2%) directly onto the Opti-TOF™ 384-well plate (Applied Biosystems, Foster City, Calif.) and re-crystallized with 0.5 μL of matrix [10 mg/mL α-Cyano-4-hydroxycinnamic acid in acetonitrile/H 2 O/TFA (70%:25%:0.2%). Peptide Map Fingerprint result-dependent MS/MS analysis is performed on a 4800 Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Time of Flight (MALDI TOF-TOF) mass spectrometer (Applied Biosystems, Foster City, Calif.). In short, peptide fingerprint is generated scanning 700-4,000 Da mass range using 1000 shots acquired from 20 randomized regions of the sample spot at 3600 intensity of OptiBeam™ on-axis laser in positive reflectron mode. Ten most abundant precursors, excluding trypsin autolysis peptides and sodium/potassium adducts, are selected for subsequent tandem MS analysis, where 2000 total shots are taken with 4200 laser intensity. Post-source decay (PSD) fragments from the precursors of interest are isolated by timed-ion selection and reaccelerated into the reflectron to generate the MS/MS spectrum. Raw data is deconvoluted using GPS Explorer™ software and submitted for peptide mapping and MS/MS ion search analysis against non-redundant NCBI database with an in-house licensed Mascot search engine (Matrix Science, London; UK). 
     MS Results: 
     1A) Pancreatin API-1&amp;2-A (API peaks 1 and 2-A) ( FIG. 22 ). Match to: gi|129436 Score: 516 Expect: 1.2e-046 Phospholipase A2, minor isoenzyme (Phosphatidylcholine 2-acylhydrolase). 
     Nominal mass (Mr): 13959, Calculated pI value: 5.61; NCBI BLAST search of gi1129436 against nr; Taxonomy:  Sus scrofa ; Cleavage by Trypsin: cuts C-term side of KR unless next residue is P; Variable modifications: Carbamidomethyl (C), Deamidation (NQ), Oxidation (M); Sequence Coverage: 85%, Score: 61-93; Sequenced and Matched peptides (Fingerprints) shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 1) 
                   
               
               
                    1  ALWQFR SMIK  CTIPGSDPLL DFNNYGCYCG LGGSGTPVDE LDRCCETHDN   
                   
               
               
                   
               
               
                   51  CYR DAK NLDS CKFLVDNPYT NSYSYSCSNT EITCNSKNNA CEAFICNCDR   
               
               
                   
               
               
                   101  NAAICFSKAP YNK EHKNLDT KKYC 
               
             
          
         
       
     
     1B) Pancreatin API-1&amp;2-B ( FIG. 22 ). Match to: gi|47523482 Score: 227 Expect: 9.7e-018 pancreatic colipase [ Sus scrofa].    
     Nominal mass (Mr): 12132, Calculated pI value: 5.29; NCBI BLAST search of gi|47523482 against nr; Taxonomy:  Sus scrofa ; Links to retrieve other entries containing this sequence from NCBI Entrez: gi|71063983 from  Sus scrofa , gi|7711136 from  Sus scrofa , gi|24418847 from  Sus scrofa ; Variable modifications: Carbamidomethyl (C), Deamidation (NQ), Oxidation (M); Cleavage by Trypsin: cuts C-term side of KR unless next residue is P; Sequence Coverage: 33%; Sequenced and Matched peptides (Fingerprints) shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 2) 
                   
               
               
                    1   MEKVLALLLV TLTVAYAVPD  PRGIIINLDE GELCLNSAQC KSNCCQHDTI   
                   
               
               
                   
               
               
                   51  LSLSRCALK A RENSECSAFT LYGVYYKCPC ERGLTCEGDK SLVGSITNTN 
               
               
                   
               
               
                   101   FGICHDVGRS SD 
               
             
          
         
       
     
     2) Pancreatin API-3 ( FIG. 23 ). Match to: gi|67161 Score: 147 Expect: 9.7e-010; triacylglycerol lipase (EC 3.1.1.3)—pig. 
     Nominal mass (M r ): 49894; Calculated pI value: 5.63; NCBI BLAST search of gi|67161 against nr; Unformatted sequence string for pasting into other applications; Taxonomy:  Sus scrofa domestica ; Variable modifications: Carbamidomethyl (C), Deamidation (NQ), Oxidation (M); Cleavage by Trypsin: cuts C-term side of KR unless next residue is P; Sequence Coverage: 18%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 3) 
                   
               
               
                    1   SEVCFPRLGC FSDDAPWAGI VQRPLKILPP DKDVDTRFLL YTNQNQNNYQ 
                   
               
               
                   
               
               
                   51   ELVADPSTIT NSNFRMDRKT RFIIHGFIDK GEEDWLSNIC K NLFKVESVN   
               
               
                   
               
               
                   101  CICVDWK GGS RTGYTQASQN IRIVGAEVAY FVEVLKSSLG YSPSNVHVIG 
               
               
                   
               
               
                   151   HSLGSHAAGE AGRRTNGTIE RITGLDPAEP CFQGTPELVR LDPSDAKFVD 
               
               
                   
               
               
                   201   VIHTDAAPII PNLGFGMSQT VGHLDFFPNG GKQMPGCQKN ILSQIVDIDG 
               
               
                   
               
               
                   251   IWEGTRDFVA CNHLRSYKYY ADSILNPDGF AGFPCDSYNV FTANK CFPCP   
               
               
                   
               
               
                   301  SEGCPQMGHY ADRFPGK TNG VSQVFYLNTG DASNFARWRY KVSVTLSGK K   
               
               
                   
               
               
                   351  VTGHILVSLF GNEGNSR QYE IYKGTLQPDN THSDEFDSDV EVGDLQKVKF 
               
               
                   
               
               
                   401   IWYNNNVINP TLPRVGASKI TVER NDGKVY DFCSQETVRE EVLLTLNPC   
               
             
          
         
       
     
     3A) Pancreatin API-4 and 5-A ( FIG. 24 ). Match to: gi|62738255 Score: 138 Expect: 7.7e-009; Chain A, Crystal Structure Of The Pig Pancreatic Alpha-Amylase Complexed With Malto-Oligosaacharide. 
     Nominal mass (M r ): 55381; Calculated pI value: 5.91; NCBI BLAST search of gi|62738255 against nr; Taxonomy:  Sus scrofa ; Links to retrieve other entries containing this sequence from NCBI Entrez: gi|15825762 from  Sus scrofa ; Sequence Coverage: 20%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 4) 
                   
               
               
                    1   XYAPQTQSGR  TSIVHLFEWR WVDIALECER  YLGPK GFGGV QVSPPNENIV   
                   
               
               
                   
               
               
                   51  VTNPSRPWWE  RYQPVSYKLC TRSGNENEFR DMVTRCNNVG VR IYVDAVIN   
               
               
                   
               
               
                   101  HMCGSGAAAG TGTTCGSYCN PGNR EFPAVP YSAWDFNDGK CKTASGGIES 
               
               
                   
               
               
                   151   YNDPYQVRDC QLVGLLDLAL EKDYVRSMIA DYLNKLIDIG VAGFRIDASK 
               
               
                   
               
               
                   201   HMWPGDIKAV LDKLHNLNTN WFPAGSRPFI FQEVIDLGGE AIKSSEYFGN 
               
               
                   
               
               
                   251   GRVTEFKYGA KLGTVVRKWS GEKMSYLKNW GEGWGFMPSD RALVFVDNHD 
               
               
                   
               
               
                   301   NQRGHGAGGS SILTFWDARL YKIAVGFMLA HPYGFTRVMS SYRWARNFVN 
               
               
                   
               
               
                   351   GEDVNDWIGP PNNNGVIKEV TINADTTCGN DWVCEHRWRE IRNMVWFR NV   
               
               
                   
               
               
                   401  VDGQPFANWW DNGSNQVAFG R GNRGFIVFN NDDWQLSSTL QTGLPGGTYC 
               
               
                   
               
               
                 451  DVISGDKVGN SCTGIKVYVS SDGTAQFSIS NSAEDPFIAI HAESKL 
               
             
          
         
       
     
     3B) Pancreatin API-4 and 5-B ( FIG. 24 ). Match to: gi|3318722 Score: 120 Expect: 4.8e-007; Chain E, Leech-Derived Tryptase Inhibitor TRYPSIN COMPLEX. 
     Nominal mass (M r ): 23457; Calculated pI value: 8.26; NCBI BLAST search of gi|3318722 against nr; Taxonomy:  Sus scrofa ; gi|9954879 from  Sus scrofa ; Sequence Coverage: 29%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 5) 
                   
               
               
                    1   IVGGYTCAAN SIPYQVSLNS GSHFCGGSLI NSQWVVSAAH CYKSRIQVRL 
                   
               
               
                   
               
               
                   51   GEHNIDVLEG NEQFINAAK I ITHPNFNGNT LDNDIMLIK L SSPATLXSRV 
               
               
                   
               
               
                   101   ATVSLPRSCA AAGTECLISG WGNTK SSGSS YPSLLQCLKA PVLSDSSCKS   
               
               
                   
               
               
                   151  SYPGQITGNM ICVGFLEGGK  DSCQGDSGGP VVCNGQLQGI VSWGYGCAQK 
               
               
                   
               
               
                   201   NKPGVYTKVC NYVNWIQQTI AAN  
               
             
          
         
       
     
     3C) Pancreatin API-4 and 5-C ( FIG. 24 ). Match to: gi|2098469 Score: 132 Expect: 3.1e-008; Porcine Pancreatic Alpha-Amylase Complexed with Acarbose. 
     Nominal mass (M r ): 55325; Calculated pI value: 5.68; NCBI BLAST search of gi|2098469 against nr; Taxonomy:  Sus scrofa ; Sequence Coverage: 15%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 6) 
                   
               
               
                    1   XYAPQTQSGR  TSIVHLFEWR WVDIALECER  YLGPK GFGGV QVSPPNENIV   
                   
               
               
                   
               
               
                   51    VTNPSRPWWE R YQPVSYKLC TRSGNENEFR DMVTRCNNVG VRIYVDAVIN 
               
               
                   
               
               
                   101   HMCGSGAAAG TGTTCGSYCN PGSREFPAVP YSAWDFNDGK CKTASGGIES 
               
               
                   
               
               
                   151   YNDPYQVRDC QLVGLLDLAL EKDYVRSMIA DYLNKLIDIG VAGFRIDASK 
               
               
                   
               
               
                   201   HMWPGDIKAV LDKLHNLNTN WFPAGSRPFI FQEVIDLGGE AIQSSEYFGN 
               
               
                   
               
               
                   251   GRVTEFKYGA KLGTVVRKWS GEKMSYLKNW GEGWGFMPSD RALVFVDNHD 
               
               
                   
               
               
                   301   NQRGHGAGGA SILTFWDARL YKVAVGFMLA HPYGFTRVMS SYRWARNFVN 
               
               
                   
               
               
                   351   GEDVNDWIGP PNNNGVIKEV TINADTTCGN DWVCEHRWRE IRNMVWFRNV 
               
               
                   
               
               
                   401   VDGEPFANWW DNGSNQVAFG RGNRGFIVFN NDDWQLSSTL QTGLPGGTYC 
               
               
                   
               
               
                   451   DVISGDKVGN SCTGIK VYVS SDGTAQFSIS NSAEDPFIAI HAESKL   
               
             
          
         
       
     
     4) Pancreatin API-6 and 7 ( FIG. 25 ). Match to: gi|136429 Score: 296 Expect: 1.2e-024. 
     Nominal mass (Mr): 24394, Calculated pI value: 7.00; Taxonomy:  Sus scrofa ; Sequence Coverage: 44%; Sequenced and Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 7) 
                   
               
               
                 
                     1  FPTDDDDKIV GGYTCAANSI PYQVSLNSGS HFCGGSLINS QWVVSAAHCY 
                 
                   
               
               
                   
               
               
                 
                    51  KSRIQVRLGE HNIDVLEGNE QFINAAKIIT HPNFNGNTLD NDIMLIKLSS 
                 
               
               
                   
               
               
                 
                   101  PATLNSRVAT VSLPRSCAAA GTECLISGWG NTKSSGSSYP SLLQCLKAPV 
                 
               
               
                   
               
               
                 
                   151  LSDSSCKSSY PGQITGNMIC VGFLEGGKDS CQGDSGGPVV CNGQLQGIVS 
                 
               
               
                   
               
               
                 
                   201  WGYGCAQKNK PGVYTKVCNY VNWIQQTIAA N 
                 
               
             
          
         
       
     
     5) Pancreatin API-8 and 9 ( FIG. 26 ). Match to: gi|47523026 Score: 405 Expect: 1.5e-035; pancreatic elastase II [ Sus scrofa].    
     Nominal mass (M r ): 28681, Calculated pI value: 8.33; NCBI BLAST search of gi|47523026 against nr; Unformatted sequence string for pasting into other applications; Taxonomy:  Sus scrofa ; Links to retrieve other entries containing this sequence from NCBI Entrez: gi|119258 from  Sus scrofa , gi|164442 from  Sus scrofa ; Variable modifications: Carbamidomethyl (C), Deamidation (NQ), Oxidation (M); Cleavage by Trypsin: cuts C-term side of KR unless next residue is P; Sequence Coverage: 54%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 8) 
                   
               
               
                    1   MIRALLLSTL VAGALSCGLP ANLPQLPR VV GGEDARPNSW PWQVSLQYDS   
                   
               
               
                   
               
               
                   51    SGQWRHTCGG TLVDQSWVLT AAHCISSSRT YR VVLGRHSL STNEPGSLAV 
               
               
                   
               
               
                   101   KVSK LVVHQD WNSNQLSNGN DIALLKLASP VSLTDKIQLG CLPAAGTILP   
               
               
                   
               
               
                   151    NNYVCYVTGW GRLQTNGASP DILQQGQLLV VDYATCSKPG WWGSTVK TNM 
               
               
                   
               
               
                   201   ICAGGDGIIS SCNGDSGGPL NCQGANGQWQ VHGIVSFGSS LGCNYYHKPS 
               
             
          
         
       
     
     6A) Pancreatin API-10-A ( FIG. 27 ). Match to: gi|67161 Score: 486 Expect: 1.2e-043; triacylglycerol lipase (EC 3.1.1.3)—pig. 
     Nominal mass (M r ): 49894; Calculated pI value: 5.63; NCBI BLAST search of gi|67161 against nr; Unformatted sequence string for pasting into other applications; Taxonomy:  Sus scrofa domestica ; Sequence Coverage: 56%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 9) 
                   
               
               
                    1  SEVCFPRLGC FSDDAPWAGI VQRPLK ILPP DKDVDTR FLL YTNQNQNNYQ   
                   
               
               
                   
               
               
                   51  ELVADPSTIT NSNFR MDRKT R FIIHGFIDK GEEDWLSNIC K NLFKVESVN 
               
               
                   
               
               
                   101   CICVDWKGGS R TGYTQASQN IRIVGAEVAY FVEVLKSSLG YSPSNVHVIG   
               
               
                   
               
               
                   151  HSLGSHAAGE AGR RTNGTIE R ITGLDPAEP CFQGTPELVR  LDPSDAKFVD 
               
               
                   
               
               
                   201   VIHTDAAPII PNLGFGMSQT VGHLDFFPNG GKQMPGCQK N ILSQIVDIDG   
               
               
                   
               
               
                   251  IWEGTRDFVA CNHLR SYKYY ADSILNPDGF AGFPCDSYNV FTANK CFPCP   
               
               
                   
               
               
                   301  SEGCPQMGHY ADRFPGKTNG VSQVFYLNTG DASNFAR WRY KVSVTLSGK K   
               
               
                   
               
               
                   351  VTGHILVSLF GNEGNSR QYE IYKGTLQPDN THSDEFDSDV EVGDLQKVKF 
               
               
                   
               
               
                   401   IWYNNNVINP TLPRVGASKI TVERNDGK VY DFCSQETVRE EVLLTLNPC   
               
             
          
         
       
     
     6b) Pancreatin API-10-B: similar to chymotrypsin-like ( FIG. 27   b ). Match to: gi|73957472 Score: 129 Expect: 6.1e-008; PREDICTED: similar to chymotrypsin-like. 
     Nominal mass (M r ): 28200; Calculated pI value: 8.85; NCBI BLAST search of gi|73957472 against nr; Taxonomy:  Canis familiaris ; Sequence Coverage: 12%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 10) 
                   
               
               
                    1   MLLLSLTLSL VLLGSSWGCG IPAIKPVLSF SQRIVNGENA VPGSWPWQVS 
                   
               
               
                   
               
               
                    51   LQDKSGFHFC GGSLISQSWV VTAAHCNVIP GRHVVVLGEY DRSSNAEPLQ 
               
               
                   
               
               
                   101   VLSISKAITY PSWNPTTLNN DLTLLKLASP ARYTQRISPV CLASPDEELP 
               
               
                   
               
               
                   151   AGLKCATTGW GR LSGVGNVT PARLQQVALP LVTVNECRQY WGSR ITDAMI 
               
               
                   
               
               
                   201   CAGGSGASSC QGDSGGPLVC QKGNTWVLIG IVSWGTTNCN VRQPAIYTRV 
               
               
                   
               
               
                   251   SKFSTWISQV IAYN 
               
             
          
         
       
     
     7A) Pancreatin API-12-A: triacylglycerol lipase (EC 3.1.1.3) ( FIG. 28 ). 
     Nominal mass (Mr): 49894; Calculated pI value: 5.63; Taxonomy:  Sus scrofa domestica ; Sequence Coverage: 62%, Score: 80-98; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 11) 
                   
               
               
                    1  SEVCFPR LGC FSDDAPWAGI VQRPLKILPP DKDVDTR FLL YTNQNQNNYQ   
                   
               
               
                   
               
               
                   51  ELVADPSTIT NSNFR MDRKT R FIIHGFIDK GEEDWLSNIC K NLFKVESVN 
               
               
                   
               
               
                   101   CICVDWKGGS R TGYTQASQN IR IVGAEVAY FVEVLK SSLG YSPSNVHVIG   
               
               
                   
               
               
                   151  HSLGSHAAGE AGR RTNGTIE R ITGLDPAEP CFQGTPELVR  LDPSDAK FVD   
               
               
                   
               
               
                   201  VIHTDAAPII PNLGFGMSQT VGHLDFFPNG GK QMPGCQK N ILSQIVDIDG   
               
               
                   
               
               
                   251  IWEGTRDFVA CNHLR SYK YY ADSILNPDGF AGFPCDSYNV FTANKCFPCP   
               
               
                   
               
               
                   301  SEGCPQMGHY ADRFPGK TNG VSQVFYLNTG DASNFARWRY KVSVTLSGK K   
               
               
                   
               
               
                   351  VTGHILVSLF GNEGNSR QYE IYKGTLQPDN THSDEFDSDV EVGDLQKVK F   
               
               
                   
               
               
                   401  IWYNNNVINP TLPR VGASKI TVER NDGKVY DFCSQETVRE EVLLTLNPC   
               
             
          
         
       
     
     7B) Pancreatin API-12-B: triacylglycerol lipase (EC 3.1.1.3) ( FIG. 28 ). 
     Nominal mass (Mr): 49894; Calculated pI value: 5.63; Taxonomy:  Sus scrofa domestica ; Sequence Coverage: 33%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 12) 
                   
               
               
                    1  SEVCFPRLGC FSDDAPWAGI VQRPLK ILPP DKDVDTR FLL YTNQNQNNYQ   
                   
               
               
                   
               
               
                   51  ELVADPSTIT NSNFR MDRKT RFIIHGFIDK GEEDWLSNIC KNLFKVESVN 
               
               
                   
               
               
                   101   CICVDWKGGS RTGYTQASQN IRIVGAEVAY FVEVLKSSLG YSPSNVHVIG 
               
               
                   
               
               
                 151  HSLGSHAAGE AGRRTNGTIE R ITGLDPAEP CFQGTPELVR  LDPSDAKFVD 
               
               
                   
               
               
                   201   VIHTDAAPII PNLGFGMSQT VGHLDFFPNG GK QMPGCQK N ILSQIVDIDG 
               
               
                   
               
               
                   251   IWEGTRDFVA CNHLRSYK YY ADSILNPDGF AGFPCDSYNV FTANK CFPCP 
               
               
                   
               
               
                   301   SEGCPQMGHY ADR FPGKTNG VSQVFYLNTG DASNFAR WRY KVSVTLSGK K   
               
               
                   
               
               
                   351  VTGHILVSLF GNEGNSR QYE IYKGTLQPDN THSDEFDSDV EVGDLQKVKF 
               
               
                   
               
               
                   401   IWYNNNVINP TLPRVGASKI TVERNDGKVY DFCSQETVRE EVLLTLNPC 
               
             
          
         
       
     
     7C) Pancreatin API-12-C ( FIG. 28 ): Lithostathine precursor ( FIG. 27   c ). Match to: gi|3024090 Score: 104 Expect: 1.9e-005. 
     Nominal mass (Mr): 13140; Calculated pI value: 4.85; Taxonomy:  Sus scrofa ; Sequence Coverage: 32%, scor: 95; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 13) 
                   
               
               
                    1   MLPSMSLPSL XWMLLSCLML LSQVQGEDSP ADTPSARISC PK GSMAYASY   
                   
               
               
                   
               
               
                   51  CYALFITPK T WMGADMACQK  RPSGHLVSVL SGAEASFVSS LIK NNLNALS 
               
               
                   
               
               
                   101   DVWIGLHDPT EGLEPNAGGW EW 
               
             
          
         
       
     
     7D) Pancreatin API-12-D ( FIG. 28 ): Chain A, The Structure Of Native Porcine Pancreatic Elastase. Match to: gi|7546312 Score: 121 Expect: 3.8e-007. 
     Nominal mass (Mr): 25904; Calculated pI value: 8.42; Sequence Coverage: 34%; Taxonomy:  Sus scrofa ; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 14) 
                   
               
               
                    1   XVGGTEAQRN SWPSQISLQY RSGSSWAHTC GGTLIRQNWV MTAAHCVDRE 
                   
               
               
                   
               
               
                   51   LTFRVVVGEH NLNQNDGTEQ YVGVQKIVVH PYWNTDDVAA GYDIALLRLA 
               
               
                   
               
               
                   101   QSVTLNSYVQ LGVLPRAGTI LANNSPCYIT GWGLTR TNGQ LAQTLQQAYL   
               
               
                   
               
               
                 
                   151  PTVDYAICSS SSYWGSTVKN SMVCAGGDGV RSGCQGDSGG PLHCLVNGQY 
                 
               
               
                   
               
               
                   201  AVHGVTSFVS R LGCNVTR KP TVFTR VSAYI SWINNVIASN  
               
             
          
         
       
     
     7E) Pancreatin API-12-E ( FIG. 28 ): Chain A, The Structure Of Native Porcine Pancreatic Elastase. Match to: gi|7546312 Score: 206 Expect: 1.2e-015. 
     Nominal mass (Mr): 25904; Calculated pI value: 8.42; Taxonomy:  Sus scrofa ; Sequence Coverage: 35%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 15) 
                   
               
               
                    1   XVGGTEAQRN SWPSQISLQY R SGSSWAHTC GGTLIRQNWV MTAAHCVDR E 
                   
               
               
                   
               
               
                   51   LTFRVVVGEH NLNQNDGTEQ YVGVQKIVVH PYWNTDDVAA GYDIALLRLA 
               
               
                   
               
               
                   101   QSVTLNSYVQ LGVLPRAGTI LANNSPCYIT GWGLTRTNGQ LAQTLQQAYL 
               
               
                   
               
               
                   151   PTVDYAICSS SSYWGSTVK N SMVCAGGDGV RSGCQGDSGG PLHCLVNGQY   
               
               
                   
               
               
                   201  AVHGVTSFVS RLGCNVTRKP TVFTR VSAYI SWINNVIASN 
               
             
          
         
       
     
     8) Pancreatin API-13 ( FIG. 29   a ): The MS spectrum is good ( FIG. 29   b ). The four most abundant peptides are selected for subsequent tandem MS analysis (see Table 8). No matching sequence is found. 
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 Peptide 
                 MS 
                 Sequence 
                 Match (bold) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 901.52 
                 QGNSLLLR (SEQ ID NO: 16) 
                   
               
               
                 2 
                 1006.48 
                 NWNEMGVR (SEQ ID NO: 17) 
                 Envelope glycoprotein 
               
               
                   
                   
                   
                 (AAV69229.1) 
               
               
                 3 
                 2468.12 
                 RDHVKDDPGIPINAGD[251.07]GERR 
                 Elastase 3B 
               
               
                   
                   
                 (SEQ ID NO: 18) 
                   
               
               
                 4 
                 2989.43 
                 WLTFCAHDHRPKVQSQNPRNGAAGEPA 
                   
               
               
                   
                   
                 (SEQ ID NO: 19) 
               
               
                   
               
             
          
         
       
     
     9A) Pancreatin API-14-A: MS results are the same as that of pancreatin API-13. 
     9B) Pancreatin API-14-B ( FIG. 30 ): Chain A, Crystal Structure Of Porcine Pancreatic Elastase Complexed With A Macroclyclic. 
     Nominal mass (Mr): 25892; Calculated pI value: 8.44; NCBI BLAST search of gi|109157401 against nr; Unformatted sequence string for pasting into other applications; gi|4930034 from  Sus scrofa ; Sequence Coverage: 52%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 20) 
                   
               
               
                    1    VVGGTEAQRN SWPSQISLQY R SGSSWAHTC GGTLIR QNWV MTAAHCVDRE   
                   
               
               
                   
               
               
                   51    LTFR VVVGEH NLNQNDGTEQ YVGVQK IVVH PYWNTDDVAA GYDIALLRLA   
               
               
                   
               
               
                   101    QSVTLNSYVQ LGVLPRAGTI LANNSPCYIT GWGLTR TNGQ LAQTLQQAYL 
               
               
                   
               
               
                   151   PTVDYAICSS SSYWGSTVK N SMVCAGGDGV R SGCQGDSGG PLHCLVNGQY 
               
               
                   
               
               
                   201   AVHGVTSFVS R LGCNVTRKP TVFTR VSAYI SWINNVIASN 
               
             
          
         
       
     
     10A) Pancreatin API-15-A ( FIG. 31 ): Chain A, The Structure Of Native Porcine Pancreatic Elastase. 
     Nominal mass (Mr): 25904; Calculated pI value: 8.42; NCBI BLAST search of gi|7546312 against nr; Taxonomy:  Sus scrofa ; Sequence Coverage: 59%, score: 84; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 21) 
                   
               
               
                    1   XVGGTEAQR N SWPSQISLQY RSGSSWAHTC GGTLIRQNWV MTAAHCVDRE   
                   
               
               
                   
               
               
                   51    LTFR VVVGEH NLNQNDGTEQ YVGVQK IVVH PYWNTDDVAA GYDIALLRLA   
               
               
                   
               
               
                   101    QSVTLNSYVQ LGVLPRAGTI LANNSPCYIT GWGLTR TNGQ LAQTLQQAYL 
               
               
                   
               
               
                   151   PTVDYAICSS SSYWGSTVKN SMVCAGGDGV R SGCQGDSGG PLHCLVNGQY   
               
               
                   
               
               
                   201    AVHGVTSFVS R LGCNVTR KP TVFTR VSAYI SWINNVIASN 
               
             
          
         
       
     
     10B) Pancreatin API-15-B ( FIG. 31 ): Chain A, The Structure Of Native Porcine Pancreatic Elastase. 
     Nominal mass (Mr): 25904; Calculated pI value: 8.42; NCBI BLAST search of gi|7546312 against nr; Taxonomy:  Sus scrofa ; Sequence Coverage: 59%, Score: 64-85; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 22) 
                   
               
               
                    1   XVGGTEAQR N SWPSQISLQY R SGSSWAHTC GGTLIR QNWV MTAAHCVDRE   
                   
               
               
                   
               
               
                   51    LTFRVVVGEH NLNQNDGTEQ YVGVQKIVVH PYWNTDDVAA GYDIALLRLA   
               
               
                   
               
               
                   101    QSVTLNSYVQ LGVLPRAGTI LANNSPCYIT GWGLTR TNGQ LAQTLQQAYL 
               
               
                   
               
               
                   151   PTVDYAICSS SSYWGSTVKN SMVCAGGDGV R SGCQGDSGG PLHCLVNGQY   
               
               
                   
               
               
                   201    AVHGVTSFVS R LGCNVTRKP TVFTRVSAYI SWINNVIASN 
               
             
          
         
       
     
     10C) Pancreatin API-15-B ( FIG. 31 ): Pancreatic colipase. 
     Nominal mass (Mr): 12132; Calculated pI value: 5.29; NCBI BLAST search of gi|47523482 against nr; gi|71063983 from  Sus scrofa ; gi|7711136 from  Sus scrofa ; gi|24418847 from  Sus scrofa ; Sequence Coverage: 70%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 23) 
                   
               
               
                    1   MEKVLALLLV TLTVAYAVPD PR GIIINLDE GELCLNSAQC KSNCCQHDTI   
                   
               
               
                   
               
               
                   51    LSLSRCALKA RENSECSAFT LYGVYYKCPC ER GLTCEGDK  SLVGSITNTN   
               
               
                   
               
               
                   101    FGICHDVGR S SD 
               
             
          
         
       
     
     11) Pancreatin API-17 ( FIG. 32 ): Chain A, The Structure Of Native Porcine Pancreatic Elastase. 
     Nominal mass (Mr): 25904; Calculated pI value: 8.42; NCBI BLAST search of gi|7546312 against nr; Taxonomy:  Sus scrofa ; Sequence Coverage: 42%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 24) 
                   
               
               
                    1   XVGGTEAQR N SWPSQISLQY R SGSSWAHTC GGTLIRQNWV MTAAHCVDRE 
                   
               
               
                   
               
               
                    51   LTFRVVVGEH NLNQNDGTEQ YVGVQK IVVH PYWNTDDVAA GYDIALLRLA   
               
               
                   
               
               
                   101    QSVTLNSYVQ LGVLPRAGTI LANNSPCYIT GWGLTR TNGQ LAQTLQQAYL 
               
               
                   
               
               
                   151   PTVDYAICSS SSYWGSTVKN SMVCAGGDGV R SGCQGDSGG PLHCLVNGQY   
               
               
                   
               
               
                   201    AVHGVTSFVS R LGCNVTRKP TVFTRVSAYI SWINNVIASN  
               
             
          
         
       
     
     12A) Pancreatin API-19-A ( FIG. 33 ): Chain A, Structure Of Porcine Pancreatic Alpha-Amylase. Match to: gi|2780980 Score: 828 Expect: 7.7e-078. 
     Nominal mass (Mr): 55310; Calculated pI value: 5.79; NCBI BLAST search of gi|2780980 against nr; Taxonomy:  Sus scrofa ; Sequence Coverage: 36%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 25) 
                   
               
               
                    1   XYAPQTQSGR  TSIVHLFEWR WVDIALECER  YLGPKGFGGV QVSPPNENVV 
                   
               
               
                   
               
               
                   51   VTNPSRPWWE RYQPVSYKLC TR SGNENEFR DMVTR CNNVG VRIYVDAVIN 
               
               
                   
               
               
                   101   HMCGSGAAAG TGTTCGSYCN PGSREFPAVP YSAWDFNDGK CK TASGGIES   
               
               
                   
               
               
                   151    YNDPYQVRDC QLVGLLDLAL EKDYVR SMIA DYLNK LIDIG VAGFR IDASK 
               
               
                   
               
               
                   201   HMWPGDIKAV LDKLHNLNTN WFPAGSRPFI FQEVIDLGGE AIQSSEYFGN 
               
               
                   
               
               
                   251   GRVTEFKYGA KLGTVVRKWS GEKMSYLK NW GEGWGFMPSD RALVFVDNHD   
               
               
                   
               
               
                   301    NQRGHGAGGA SILTFWDAR L YK VAVGFMLA HPYGFTR VMS SYRWARNFVN 
               
               
                   
               
               
                   351   GEDVNDWIGP PNNNGVIK EV TINADTTCGN DWVCEHR WRE IR NMVWFRNV   
               
               
                   
               
               
                   401    VDGQPFANWW DNGSNQVAFG R GNRGFIVFN NDDWQLSSTL QTGLPGGTYC 
               
               
                   
               
               
                   451   DVISGDKVGN SCTGIKVYVS SDGTAQFSIS NSAEDPFIAI HAESKL 
               
             
          
         
       
     
     12B) Pancreatin API-19-B ( FIG. 33 ): Chain A, Structure Of Porcine Pancreatic Alpha-Amylase. 
     Nominal mass (Mr): 55310; Calculated pI value: 5.79; NCBI BLAST search of gi|2780980 against nr; Unformatted sequence string for pasting into other applications; Taxonomy:  Sus scrofa ; Sequence Coverage: 29%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 26) 
                   
               
               
                    1   XYAPQTQSGR TSIVHLFEWR WVDIALECER YLGPKGFGGV QVSPPNENVV 
                   
               
               
                   
               
               
                   51   VTNPSRPWWE RYQPVSYK LC TRSGNENEFR  DMVTRCNNVG VRIYVDAVIN 
               
               
                   
               
               
                   101   HMCGSGAAAG TGTTCGSYCN PGSREFPAVP YSAWDFNDGK CK TASGGIES   
               
               
                   
               
               
                   151    YNDPYQVRDC QLVGLLDLAL EKDYVR SMIA DYLNK LIDIG VAGFR IDASK 
               
               
                   
               
               
                   201   HMWPGDIKAV LDKLHNLNTN WFPAGSRPFI FQEVIDLGGE AIQSSEYFGN 
               
               
                   
               
               
                   251   GRVTEFKYGA KLGTVVRKWS GEKMSYLK NW GEGWGFMPSD R ALVFVDNHD 
               
               
                   
               
               
                   301   NQR GHGAGGA SILTFWDAR L YK VAVGFMLA HPYGFTR VMS SYRWARNFVN 
               
               
                   
               
               
                   351   GEDVNDWIGP PNNNGVIK EV TINADTTCGN DWVCEHR WRE IR NMVWFRNV   
               
               
                   
               
               
                   401    VDGQPFANWW DNGSNQVAFG R GNRGFIVFN NDDWQLSSTL QTGLPGGTYC 
               
               
                   
               
               
                   451   DVISGDKVGN SCTGIKVYVS SDGTAQFSIS NSAEDPFIAI HAESKL 
               
             
          
         
       
     
     12C) Pancreatin API-19-C ( FIG. 33 ): alpha-amylase [ Sus scrofa].    
     Nominal mass (Mr): 57050; Calculated pI value: 6.51; NCBI BLAST search of gi|47523476 against nr; Taxonomy:  Sus scrofa ; Sequence Coverage: 45%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 27) 
                   
               
               
                    1   MKLFLLLSAF GFCWAQYAPQ TQSGRTSIVH LFEWRWVDIA LECERYLGPK 
                   
               
               
                   
               
               
                   51   GFGGVQVSPP NENIVVTNPS RPWWERYQPV SYKLCTR SGN ENEFRDMVTR   
               
               
                   
               
               
                   101   CNNVGVR IYV DAVINHMCGS GAAAGTGTTC GSYCNPGNRE FPAVPYSAWD   
               
               
                   
               
               
                   151    FNDGK CK TAS GGIESYNDPY QVRDCQLVGL LDLALEKDYV R SMIADYLNK 
               
               
                   
               
               
                   201    LIDIGVAGFR  IDASKHMWPG DIKAVLDKLH NLNTNWFPAG SRPFIFQEVI 
               
               
                   
               
               
                   251   DLGGEAIQSS EYFGNGRVTE FKYGAKLGTV VRKWSGEKMS YLK NWGEGWG   
               
               
                   
               
               
                   301    FMPSDRALVF VDNHDNQRGH GAGGASILTF WDAR LYK VAV GFMLAHPYGF   
               
               
                   
               
               
                   351    TR VMSSYRWA R NFVNGQDVN DWIGPPNNNG VIKEVTINAD TTCGNDWVCE   
               
               
                   
               
               
                   401    HRWR QIR NMV WFRNVVDGQP FANWWANGSN QVAFGR GNRG FIVFNNDDWQ 
               
               
                   
               
               
                   451   LSSTLQTGLP GGTYCDVISG DKVGNSCTGI KVYVSSDGTA QFSISNSAED 
               
               
                   
               
               
                   501   PFIAIHAESK L  
               
             
          
         
       
     
     13) Pancreatin API-21 ( FIG. 34 ): Chain A, Structure Of Porcine Pancreatic Alpha-Amylase. Match to: gi|2780980 Score: 285 Expect: 1.5e-023. 
     Nominal mass (Mr): 55310; Calculated pI value: 5.79; NCBI BLAST search of gi|2780980 against nr; Unformatted sequence string for pasting into other applications; Taxonomy:  Sus scrofa ; Sequence Coverage: 31%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 28) 
                   
               
               
                    1   XYAPQTQSGR TSIVHLFEWR WVDIALECER YLGPKGFGGV QVSPPNENVV 
                   
               
               
                   
               
               
                   51   VTNPSRPWWE RYQPVSYKLC TRSGNENEFR DMVTRCNNVG VRIYVDAVIN 
               
               
                   
               
               
                   101   HMCGSGAAAG TGTTCGSYCN PGSREFPAVP YSAWDFNDGK CK TASGGIES   
               
               
                   
               
               
                   151    YNDPYQVRDC QLVGLLDLAL EKDYVR SMIA DYLNK LIDIG VAGFR IDASK 
               
               
                   
               
               
                   201   HMWPGDIKAV LDKLHNLNTN WFPAGSRPFI FQEVIDLGGE AIQSSEYFGN 
               
               
                   
               
               
                   251   GRVTEFKYGA KLGTVVRKWS GEKMSYLK NW GEGWGFMPSD R ALVFVDNHD 
               
               
                   
               
               
                   301   NQR GHGAGGA SILTFWDAR L YK VAVGFMLA HPYGFTR VMS SYR WARNFVN   
               
               
                   
               
               
                   351    GEDVNDWIGP PNNNGVIKEV TINADTTCGN DWVCEHR WRE IRNMVWFR NV   
               
               
                   
               
               
                   401    VDGQPFANWW DNGSNQVAFG R GNRGFIVFN NDDWQLSSTL QTGLPGGTYC 
               
               
                   
               
               
                   451   DVISGDKVGN SCTGIKVYVS SDGTAQFSIS NSAEDPFIAI HAESKL 
               
             
          
         
       
     
     14) Pancreatin API-22 ( FIG. 35 ): Carboxypeptidase B. Match to: gi|5457422 Score: 239 Expect: 6.1e-019. 
     Nominal mass (M r ): 47351; Calculated pI value: 5.24; NCBI BLAST search of gi|5457422 against nr; Taxonomy:  Sus scrofa ; Links to retrieve other entries containing this sequence from NCBI Entrez: gi|47523424 from  Sus scrofa , gi|62906849 from  Sus scrofa ; Sequence Coverage: 43%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 29) 
                   
               
               
                    1   MLAFLILVTV TLASAHHSGE HFEGEKVFRV NVEDENDISL LHELASTRQI 
                   
               
               
                   
               
               
                   51   DFWKPDSVTQ IKPHSTVDFR VKAEDILAVE DFLEQNELQY EVLINNLRSV 
               
               
                   
               
               
                   101   LEAQFDSRVR TTGHSYEK YN NWETIEAWTK  QVTSENPDLI SRT AIGTTFL   
               
               
                   
               
               
                   151    GNNIYLLKVG KPGPNKPAIF MDCGFHAREW ISHAFCQWFV REAVLTYGYE   
               
               
                   
               
               
                   201    SHMTEFLNK L DFYVLPVLNI DGYIYTWTKN RMWRK TRSTN AGTTCIGTDP   
               
               
                   
               
               
                   251    NR NFDAGWCT TGASTDPCDE TYCGSAAESE KETK ALADFI R NNLSSIK AY   
               
               
                   
               
               
                   301    LTIHSYSQMI LYPYSYDYK L PENNAELNNL AKAAVKELAT LYGTK YTYGP   
               
               
                   
               
               
                   351    GATTIYPAAG GSDDWAYDQG IKYSFTFELR  DKGR YGFILP ESQIQATCEE   
               
               
                   
               
               
                   401    TMLAIK YVTN YVLGHL  
               
             
          
         
       
     
     15A) Pancreatin API-23-A ( FIG. 36 ): alpha-amylase. Match to: gi|47523476 Score: 589 Expect: 6.1e-054; alpha-amylase [ Sus scrofa].    
     Nominal mass (M r ): 57050; Calculated pI value: 6.51; NCBI BLAST search of gi|47523476 against nr; Taxonomy:  Sus scrofa ; Links to retrieve other entries containing this sequence from NCBI Entrez: gi|6056338 from  Sus scrofa , gi|33860120 from  Sus scrofa ; Sequence Coverage: 44%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 30) 
                   
               
               
                    1    MKLFLLLSAF GFCWAQYAPQ TQSGRTSIVH LFEWRWVDIA LECER YLGPK 
                   
               
               
                   
               
               
                   51    GFGGVQVSPP NENIVVTNPS RPWWER YQPV SYKLCTRSGN ENEFRDMVTR 
               
               
                   
               
               
                   101   CNNVGVR IYV DAVINHMCGS GAAAGTGTTC GSYCNPGNR E FPAVPYSAWD 
               
               
                   
               
               
                   151   FNDGKCK TAS GGIESYNDPY QVRDCQLVGL LDLALEKDYV R SMIADYLNK 
               
               
                   
               
               
                   201    LIDIGVAGFR  IDASKHMWPG DIKAVLDKLH NLNTNWFPAG SRPFIFQEVI 
               
               
                   
               
               
                   251   DLGGEAIQSS EYFGNGRVTE FKYGAKLGTV VRKWSGEKMS YLK NWGEGWG   
               
               
                   
               
               
                   301    FMPSDRALVF VDNHDNQRGH GAGGASILTF WDAR LYK VAV GFMLAHPYGF   
               
               
                   
               
               
                   351    TR VMSSYRWA RNFVNGQDVN DWIGPPNNNG VIK EVTINAD TTCGNDWVCE   
               
               
                   
               
               
                   401    HR WRQIR NMV WFR NVVDGQP FANWWANGSN QVAFGRGNRG FIVFNNDDWQ 
               
               
                   
               
               
                   451   LSSTLQTGLP GGTYCDVISG DKVGNSCTGI KVYVSSDGTA QFSISNSAED 
               
               
                   
               
               
                   501   PFIAIHAESK L 
               
             
          
         
       
     
     15B) Pancreatin API-23-B ( FIG. 36 ): Chain A, Porcine Pancreatic Alpha-Amylase. Match to: gi|2780980 Score: 712 Expect: 3.1e-066; Chain A, Structure Of Porcine Pancreatic Alpha-Amylase. 
     Nominal mass (M r ): 55310; Calculated pI value: 5.79; NCBI BLAST search of gi|2780980 against nr; Unformatted sequence string for pasting into other applications; Taxonomy:  Sus scrofa ; Variable modifications: Carbamidomethyl (C), Deamidation (NQ), Oxidation (M); Cleavage by Trypsin: cuts C-term side of KR unless next residue is P; Sequence Coverage: 35%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 31) 
                   
               
               
                    1   XYAPQTQSGR TSIVHLFEWR WVDIALECER YLGPKGFGGV QVSPPNENVV 
                   
               
               
                   
               
               
                   51   VTNPSRPWWE RYQPVSYKLC TR SGNENEFR DMVTR CNNVG VRIYVDAVIN 
               
               
                   
               
               
                   101   HMCGSGAAAG TGTTCGSYCN PGSR EFPAVP YSAWDFNDGK  CK TASGGIES   
               
               
                   
               
               
                   151    YNDPYQVRDC QLVGLLDLAL EKDYVR SMIA DYLNK LIDIG VAGFR IDASK 
               
               
                   
               
               
                   201   HMWPGDIKAV LDKLHNLNTN WFPAGSRPFI FQEVIDLGGE AIQSSEYFGN 
               
               
                   
               
               
                   251   GRVTEFKYGA KLGTVVRKWS GEKMSYLK NW GEGWGFMPSD RALVFVDNHD   
               
               
                   
               
               
                   301    NQRGHGAGGA SILTFWDAR L YK VAVGFMLA HPYGFTR VMS SYRWARNFVN 
               
               
                   
               
               
                   351   GEDVNDWIGP PNNNGVIK EV TINADTTCGN DWVCEHR WRE IR NMVWFRNV   
               
               
                   
               
               
                   401    VDGQPFANWW DNGSNQVAFG R GNRGFIVFN NDDWQLSSTL QTGLPGGTYC 
               
               
                   
               
               
                   451   DVISGDKVGN SCTGIKVYVS SDGTAQFSIS NSAEDPFIAI HAESKL 
               
             
          
         
       
     
     15C) Pancreatin API-23-C ( FIG. 36 ): Chain A, Structure Of Porcine Pancreatic Alpha-Amylase. Match to: gi|2780980 Score: 539 Expect: 6.1e-049; Chain A, Structure Of Porcine Pancreatic Alpha-Amylase. 
     Nominal mass (M r ): 55310; Calculated pI value: 5.79; NCBI BLAST search of gi|2780980 against nr; Unformatted sequence string for pasting into other applications; Taxonomy:  Sus scrofa ; Variable modifications: Carbamidomethyl (C), Deamidation (NQ), Oxidation (M); Cleavage by Trypsin: cuts C-term side of KR unless next residue is P; Sequence Coverage: 37%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 32) 
                   
               
               
                    1   XYAPQTQSGR  TSIVHLFEWR WVDIALECER YLGPK GFGGV QVSPPNENVV 
                   
               
               
                   
               
               
                   51   VTNPSRPWWE RYQPVSYKLC TR SGNENEFR DMVTR CNNVG VRIYVDAVIN 
               
               
                   
               
               
                   101   HMCGSGAAAG TGTTCGSYCN PGSREFPAVP YSAWDFNDGK CK TASGGIES   
               
               
                   
               
               
                   151    YNDPYQVRDC QLVGLLDLAL EKDYVR SMIA DYLNK LIDIG VAGFR IDASK 
               
               
                   
               
               
                   201   HMWPGDIKAV LDKLHNLNTN WFPAGSRPFI FQEVIDLGGE AIQSSEYFGN 
               
               
                   
               
               
                   251   GRVTEFKYGA KLGTVVRKWS GEKMSYLK NW GEGWGFMPSD RALVFVDNHD   
               
               
                   
               
               
                   301    NQRGHGAGGA SILTFWDAR L YK VAVGFMLA HPYGFTR VMS SYRWARNFVN 
               
               
                   
               
               
                   351   GEDVNDWIGP PNNNGVIK EV TINADTTCGN DWVCEHR WRE IR NMVWFRNV   
               
               
                   
               
               
                   401    VDGQPFANWW DNGSNQVAFG R GNRGFIVFN NDDWQLSSTL QTGLPGGTYC 
               
               
                   
               
               
                   451   DVISGDKVGN SCTGIKVYVS SDGTAQFSIS NSAEDPFIAI HAESKL 
               
             
          
         
       
     
     15D) Pancreatin API-23-D ( FIG. 36 ): Carboxypeptidase A1. Match to: gi|47523568 Score: 653 Expect: 2.4e-060; carboxypeptidase A1 precursor [ Sus scrofa].    
     Nominal mass (M r ): 47206; Calculated pI value: 5.13; NCBI BLAST search of gi|47523568 against nr; Unformatted sequence string for pasting into other applications; Taxonomy:  Sus scrofa ; Links to retrieve other entries containing this sequence from NCBI Entrez: gi|4336196 from  Sus scrofa , gi|38258878 from  Sus scrofa ; Variable modifications: Carbamidomethyl (C), Deamidation (NQ), Oxidation (M); Cleavage by Trypsin: cuts C-term side of KR unless next residue is P; Sequence Coverage: 38%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 33) 
                   
               
               
                    1   MWGLLIFSVL LGGVLAKEDF VGHQVLRISV DDEAQVQKVK ELEDLEHLQL 
                   
               
               
                   
               
               
                   51   DFWRGPARPG FPIDVRVPFP SIQAVKVFLE AHGIRYTIMI EDVQLLLDEE 
               
               
                   
               
               
                   101   QEQMFASQGR ARTTSTFNYA TYHTLEEIYD FMDILVAEHP QLVSK LQIGS   
               
               
                   
               
               
                   151    SYEGRPIYVL KFSTGGNNRP AIWIDTGIHS REWVTQASGV WFAK KITEDY 
               
               
                   
               
               
                   201   GQDPAFTAIL DNLDIFLEIV TNPDGFAFTH SENRMWRKTR SR TSGSFCVG   
               
               
                   
               
               
                   251    VDPNRNWDAG FGGAGASSNP CSETYHGKFP NSEVEVKSIV DFVNDHGNIK   
               
               
                   
               
               
                   301    AFISIHSYSQ LLLYPYGYK T EAPADKDELD QISKSAVAAL TSLYGTK FQY   
               
               
                   
               
               
                   351    GSIITTIYQA SGGTIDWTYN QGIKYSFSFE LR DTGRYGFL LPASQIIPTA 
               
               
                   
               
               
                   401   QETWLALLTI MEHTLNHPY  
               
             
          
         
       
     
     15E) Pancreatin API-23-E ( FIG. 36 ). Match to: gi|355937 Score: 286 Expect: 1.2e-023; elastase 1, pancreatic. 
     Nominal mass (M r ): 28865; Calculated pI value: 8.42; NCBI BLAST search of gi|355937 against nr; Unformatted sequence string for pasting into other applications; Taxonomy:  Sus scrofa domestica ; Sequence Coverage: 38%; Matched peptides shown in bold. 
     
       
         
               
               
             
           
               
                 (SEQ ID NO: 34) 
                   
               
               
                    1   MLRLLVVASL VLYGHSTQDF PETNARVVGG TEAQR NSWPS QISLQYR SGS 
                   
               
               
                   
               
               
                   51   SWAHTCGGTL IRQNWVMTAA HCVDRELTFR VVVGEHNLNQ NDGYEQYVGV 
               
               
                   
               
               
                   101   QKIVVHPYWN TDDVAAGYDI ALLRLAQSVT LNSYVQLGVL PRAGTILANN 
               
               
                   
               
               
                   151   SPCYITGWGL TRTNGQLAQT LQQAYLPTVD YAICSSSSYW GSTVKNSMVC 
               
               
                   
               
               
                   201   AGGDGVR SGC QGDSGGPLHC LVNGQYAVHG VTSFVSR LGC NVTRKPTVFT 
               
               
                   
               
               
                   251   RVSAYISWIN NVIASN 
               
             
          
         
       
     
     The disclosures of all articles and references, including patents, are incorporated herein by reference. The invention and the manner and process of making and using it are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. All references cited in this specification are incorporated herein by reference. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the spirit or scope of the present invention.