Abstract:
A method for isolating derivatives of general O-linked amino acids including derivatives of O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide and of O-di-phospho serine linked bovine submaxillary mucin disaccharide is provided. These derivatives are isolated and analyzed by further enzymatically separating the N-linked oligosaccharides from the O-linked oligosaccharides and then cleave O-linked oligosaccharides by decreasing the pH levels such that de-amidations are allowed. This de-amidation results in individual O-linked glycan amino acid components removed from the original glycoprotein. The O-linked glycan amino acid components are thus isolated from the glycoprotein, and can be individually analyzed.

Description:
BACKGROUND OF THE INVENTION 
     This invention relates to methods of isolating and analyzing oligosaccharides from glycoproteins. More specifically, this invention relates to laboratory methods that can be used in order to isolate derivatives of O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide and of O-di-phospho serine linked bovine submaxillary mucin disaccharide and O-linked glycan amino acids generally. O-linked oligosaccharides are oligosaccharides that are covalently attached to serine, threonine, or very rarely tyrosine residues on a glycoprotein. 
     Cancer is a disease that has a major impact on societies across the globe. According to the  National Cancer Institute  (2015), an estimated 1,658,370 new cases of cancer will be diagnosed and 589,430 people will die from the disease this year in the United States alone. As a result, researchers and their institutions are in a continuous search for substances that are effective in promoting anti-cancer biological activity—whether the substances prevent the onset of cancer or, alternatively, slow down or stop the growth of cancer. 
     Recent scientific developments by Guo et al.,  Curr. Opin. Chem Biol . (2009) 13 5-6, and by Lindhorst,  Bielstein J. Org. Chem . (2012) 8 804-818, have shown the utility of O-linked glycan amino acids such as those linked to serine, threonine, and very rarely tyrosine as potential cancer vaccines and synthetic glycopeptides for use in general immunostimulants. Thus, a principal objective of the present invention is to provide a method for isolating O-linked oligosaccharides in order to determine the oligosaccharide anti-cancer potential. 
     Despite advances in the art, problems still remain. Many obstacles arise when isolating the oligosaccharides from glycoproteins that in turn limit the understanding of oligosaccharide anti-cancer activity. In particular, problems exist with isolating O-linked glycan amino acids with specificity. Current methods require the use of reductive methods that are both costly and stifle the depth of research because these methods and systems are not as sensitive or stable. For instance, in U.S. Patent Publication 2007/0105179 A1 discloses separating and detecting N- and O-linked oligosaccharides in glycoproteins via non-degradative enzymatic cleavage. This method does not provide for the identification and isolation of particular O-linked glycan amino acids—such as derivatives of O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide and of O-di-phospho serine linked bovine submaxillary mucin disaccharide and for general O-linked glycan amino acids—in glycoproteins that are isolated using the method disclosed herein. Therefore, a need in the art exists to address these deficiencies. 
     Thus, a primary object of the invention is to provide a method and system that improves upon the state of the art. 
     Another object of the invention is to provide a method that is simple and maximizes a preferred pH stability required to specifically isolate an O-linked glycan amino acid. 
     These and other objects, features, or advantages of the present invention will become apparent from the specification, drawings, and claims. 
     SUMMARY OF THE INVENTION 
     A method of isolating derivatives of O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide and of O-di-phospho serine linked serine linked bovine submaxillary mucin disaccharide and for O-linked glycan amino acids in general. The method includes the step of treating a glycoprotein sample by known methods in order to cleave and separate N- and O-linked oligosaccharides. Further, the treated glycoprotein is removed from the cation exchange resin in the ammonium form by adding a predetermined amount of ammonium hydroxide solution. The method also includes the steps of adding the removed sample to a tube with a predetermined amount of water. Also included in the method is adding a predetermined amount of sodium borohydride to make the corresponding alditol and removing any remaining asparagine linked oligosaccharides. The method includes the step of allowing the solution to stand at ambient temperature and capped for a predetermined amount of time. For example, a capped time period of 4 hours would be at ratio with 18 hours of uncapped time period. Additionally, the method involves evaporating the contents of the tube to an approximate volume. In one embodiment, the volume is 0.2 mL. The method then includes the steps of adding a volume of water to the remainder of the solution and passing the solution through a sodium form cation exchange resin with washing an additional amount of water before freezing the resulting solution. Lastly, the method includes preparing and analyzing the solution to determine the isolation success during the method. 
     In an alternative embodiment of the invention, the method includes the step of treating a glycoprotein sample by known methods in order to cleave and separate N- and O-linked oligosaccharides. Further, the treated glycoprotein is removed from the cation exchange resin in the ammonium form by adding a predetermined amount of ammonium hydroxide solution. The method also includes the steps of then adding the treated sample to a tube with a predetermined amount of water. Also included in the method is adding a predetermined amount of sodium borohydride to make the corresponding alditol. The method then includes eluting the first solution from the ammonium cartridge used to treat the sample by adding a predetermined amount of ammonium hydroxide solution. The method includes the step of allowing the solution to stand at ambient temperature and capped for a predetermined amount of time. For example, a capped time period of 4 hours would be at ratio with 18 hours of uncapped time period. The solution is then evaporated for an approximate amount of time. In one embodiment, this volume is 0.2 mL. The method includes adding a predetermined amount of sodium borohydride solution. In one embodiment, the sodium borohydride solution volume is 3 μL and concentration 4N. 
     The method includes the steps of injecting the treated sample on to a column read by an analysis device. Also, the method includes the step of collecting the solution containing the compound located at the chromatographic first peak by placing the analysis device tubing into a separate vial. Further, the method includes the step of collecting the solution containing the compound located at the chromatographic second peak by placing the analysis device tubing into another separate vial. The method includes the steps of pushing the contents of each vial through a separate ammonium form cation exchange cartridge and collecting in a tube of predetermined volume, evaporating the contents to a predetermined volume, adding a predetermined amount of water and pushing the solution through a sodium ion cation exchange resin. Lastly, the method includes the steps of freezing or analyzing the resultant solution. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  depicts a perspective view of a system to isolate an O-linked oligosaccharide; 
         FIG. 2  depicts a flow diagram of a method of isolating O-linked glycan amino acids from a glycoprotein; 
         FIG. 3  depicts a flow diagram of a method of removing N-linked and O-linked oligosaccharides from a glycoprotein; 
         FIG. 4  is a flow diagram of a method of isolating O-linked glycan amino acids from a glycoprotein; 
         FIG. 5  depicts mass spectrum from the larger of two peaks resulting from HPAEC-PAD; 
         FIG. 6  depicts a structure consistent with O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide; 
         FIG. 7  depicts a mass spectrum obtained using a method of isolating O-linked glycan amino acids from a glycoprotein; and 
         FIG. 8  depicts a structure consistent with O-di-phospho serine linked serine linked bovine submaxillary mucin. 
     
    
    
     DETAILED DESCRIPTION 
     Referring to the figures,  FIG. 1  depicts a system  10  utilized in order to isolate and analyze derivatives of O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide and of O-di-phospho serine linked bovine submaxillary mucin disaccharide and for O-linked glycan amino acids generally. The system includes a test tube  12 , a plurality of pipets  14 , ion exchange cartridges  16  including one in the ammonium form  18  and one in the sodium form  20 , a plurality of beakers  22 , an elution column  24 , and a freezer  26 . In addition, the system  10  in  FIG. 1  can include one or more analysis devices  28  such as a mass spectrometer (MS), or a high performance anion exchange chromatograph with pulse amperometric detection (HPAEC-PAD). 
     The method as shown in  FIG. 2 , using the system of  FIG. 1 , begins as step  100  by providing a sample  30 . In one embodiment, the sample  30  is a glycoprotein bovine submaxillary mucin. In another embodiment, the sample  30  is kappa casein. In one exemplary embodiment the sample  30  has a mass of 0.3 mg and is mixed with 1.0 mL of water in a 1.5 mL test tube  12 . At step  110 , the sample  30  is treated by known methods, such as those disclosed by Madson in U.S. Patent Publication 2007/0105179 A1. As depicted in  FIG. 3 , the sample  30  is treated to enzymatically separate N- and O-linked oligosaccharides to form a first solution  32 . In an exemplary arrangement, the sample is treated with PNGase F  34  and then sodium borohydride  36  (NaBH 4 ). 
     At step  120 , a predetermined amount of the first solution  32  is passed through the ammonium form cation exchange resin  18 . A second solution  38  is removed from the resin by adding a predetermined amount of ammonium hydroxide solution at step  130 . In one embodiment, the ammonium hydroxide solution is 2N NH 4 OH. Additionally, in a preferred embodiment the amount of ammonium hydroxide solution added is 1.0 mL. Next, at step  140 , a predetermined amount of sodium borohydride  36  is pipetted into the second solution  38  to form the third solution  40 . In one arrangement, 3.0 μL solution of sodium borohydride  36  is added, which in one embodiment is 4N solution of sodium borohydride  36 . 
     The third solution  40  is allowed to stand at ambient temperature, capped, for a predetermined amount of time at step  150 . In one embodiment, the time period is four hours. At step  160 , the third solution  40  is then uncapped and allowed to stand at ambient temperature for a predetermined amount of time. For instance, if capped time is four hours, the uncapped time is eighteen hours. 
     After the predetermined amount of time has elapsed, the third solution  40  is evaporated at step  170 . In one embodiment, the third solution  40  is evaporated until approximately 0.2 mL of the third solution  40  is left. Next at step  180 , the remainder of the third solution  40  is mixed with water  42  and passed through the sodium form cation exchange resin  20  and collecting a fourth solution or resulting solution  44  off of the ion exchange cartridge  13 . In one embodiment, approximately 1.0 mL water  42  is added. 
     At step  190 , the fourth solution  44  is placed in a freezer  26  until thawed for further analysis at step  200 . Thawing can be accomplished by placing the resulting fourth solution  44  in one of the beakers  22  filled with water  42 . In one embodiment, the fourth solution  44  is analyzed by infusion into an API Triple Quadrapole MS. In another embodiment, the fourth solution  44  is analyzed by HPAEC-PAD using an MA1 column with 310 mM NaOH and a flow rate of 0.2 mL per mintue with a borate column inserted before the analytical MA1 column. As shown in  FIG. 6 , the structure consistent with O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide is identified. 
     In an alternative embodiment, the above method can be instituted with slight variation. The method as shown in  FIG. 4 , using the system of  FIG. 1 , begins at step  200  by providing the sample  30 A. In one embodiment, the sample  30 A is a glycoprotein bovine submaxillary mucin. In another embodiment, the sample  30 A is kappa casein. At step  210 , the sample  30 A is treated by known methods, such as those depicted in  FIG. 3 . In an exemplary arrangement, the sample is treated with PNGase F  34  and then sodium borohydride  36 . At step  220 , the remaining first solution  32 A is passed through the ammonium form cation exchange resin  18 . At step  230 , a predetermined amount of the first solution  32 A is evaporated. In one embodiment, the first solution  32 A is evaporated to 0.2 mL remaining. Additionally at step  230 , the tube  12  is then capped for a predetermined amount of time and then uncapped for a predetermined amount of time. In one embodiment, the tube  12  is left capped for 4 hours and left uncapped for 18 hours. A second solution  38 A is removed from the resin by adding a predetermined amount of ammonium hydroxide solution at step  240 . In one embodiment, the ammonium hydroxide solution is 2N NH 4 OH. Additionally, in a preferred embodiment, the amount of ammonium hydroxide solution added is 1.0 mL. 
     At step  250 , a predetermined amount of sodium borohydride  36  is added, and in one arrangement water is also added to form a third solution. In one arrangement, the amount of sodium borohydride  36  added is 3 microliters, which in one embodiment is 4N solution of sodium borohydride  36 . The third solution  38 A is injected onto an elution column  24  at step  260 , which in one arrangement is a Thermo Fisher MA1 elution column. In one preferred embodiment, the third solution injected onto the column is approximately 0.01 mL. A fourth solution  40 A, located at the first chromatographic peak, is monitored and collection from the elution column  24  and deposited in one of the test tubes  12 . The fourth solution  40 A and a fifth solution  44 A collected from the solution column  24  at the second chromatographic peak are pushed through separate ammonium form a cation exchange cartridges  18  to form a sixth solution  124  resulting from the fourth solution  40 A and a seventh solution  48  resulting from the fifth solution  44 , collected in separated tubes  12  at step  280 . 
     The sixth solution  46  and the seventh solution  48  are evaporated at step  290 . In one embodiment, the sixth solution  46  and the seventh solution  48  solution are evaporated to approximately 0.2 mL and 1.0 mL of water is subsequently added. At step  300 , the sixth solution  46  and the seventh solution  48  are pushed through separate sodium ion cation exchange resins  20  to form an eighth solution  50  and a ninth solution  52 . The eighth solution  50  and the ninth solution  52  are placed in a freezer  26  at step  310  until thawed for further analysis at step  320 . In one embodiment, analysis is completed by the analysis device  28 , such as in one embodiment, an API MS by infusion onto a Triple Quadrapole MS is used. In other embodiments, analysis is completed by MALDI-TOF MS or by HPAEC-PAD. As shown in  FIG. 7 , the structure consistent with O-di-phospho serine linked bovine submaxillary mucin disaccharide is identified. 
     Previous methods provide for separating cleaved-off N-linked oligosaccharides from cleaved-off O-linked oligosaccharides, such that the cleaved-off N-linked and O-linked oligosaccharides can be separately detected as shown in  FIG. 3 . Typically, these systems require degradative steps during cleavage without specifically isolating an O-linked glycan amino acid such as O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide or O-di-phospho serine linked bovine submaxillary mucin disaccharide. 
     By contrast, the methods disclosed herein provide for isolating and analyzing O-linked oligosaccharides at a predetermined pH in order to maximize de-amidation, leaving only the derived amino acid linked glycan for analysis as depicted by  FIGS. 6 and 8 . The pH drops to a low range, in one exemplary embodiment to pH 8-9, which changes the chemistry to allow de-amidation, leaving the amino acid linked glycan. Further, the method disclosed herein allows hydrolysis of the amide bonds, both peptide derived and oligosaccharide derived which result in an isolated glycan-amino acid molecule as shown in  FIGS. 6 and 8 . This isolation and allowance has not been performed by previously documented methods. 
     By “glycoprotein” is meant a protein-oligosaccharide compound where the protein and oligosaccharide portion are covalently linked. As used herein, the protein portion of the glycoprotein includes at least one covalently attached amino acid. The protein portion can be made up of naturally occurring amino acids or non-naturally occurring amino acids. By “oligosaccharide” is meant at least two monosaccharide sugars covalently linked together. Oligosaccharides in glycoproteins are generally N-linked oligosaccharides and O-linked oligosaccharides. By “O-linked oligosaccharide” is meant an oligosaccharide covalently attached to a serine, threonine or tyrosine residue of a glycoprotein. Exemplary glycoproteins include kappa casein, thyroglobulin, the secretions of mucous membranes (i.e. mucins), and fetuin. 
     The methods herein are preferably performed in aqueous liquid solutions. Further, the methods described herein preferably maintain the stability of the O-linked oligosaccharides such that they can be readily detected for subsequent analysis. 
     Therefore, methods of isolating and analyzing oligosaccharides from glycoproteins that are simple and maximize a preferred pH stability required to specifically isolate an O-linked glycan amino acid and improves upon the art have been provided. 
     From the above discussion and accompanying figures and claims it will be appreciated that the methods of isolating and analyzing oligosaccharides from glycoproteins offer many advantages over the prior art. It will also be appreciated by those skilled in the art that other modifications could be made without parting from the spirit and scope of the invention and fall within the scope of the claims and are intended to be covered thereby.