Methods and products related to the improved analysis of carbohydrates

Abstract

The invention relates, in part, to the improved analysis of carbohydrates. In particular, the invention relates to the analysis of carbohydrates, such as N-glycans and O-glycans found on proteins and saccharides attached to lipids. Improved methods, therefore, for the study of glycosylation patterns on cells, tissue and body fluids are also provided. Information from the analysis of glycans, such as the glycosylation patterns on cells, tissues and in body fluids, can be used in diagnostic and treatment methods as well as for facilitating the study of the effects of glycosylation/altered glycosylation. Such methods are also provided. Methods are further provided to assess production processes, to assess the purity of samples containing glycoconjugates, and to select glycoconjugates with the desired glycosylation.

1 . A method of analyzing a sample containing one or more glycoconjugates, which comprise one or more glycans conjugated to a non-saccharide component, comprising: 
 (a) analyzing the glycoconjugates to characterize the glycoconjugates,    (b) analyzing the non-saccharide components of the glycoconjugates to characterize the non-saccharide components,    (c) separating the glycans from the sample containing one or more glycoconjugates,    (d) analyzing the glycans to characterize the glycans, and    (e) determining the identity and quantity of all of the glycoforms of the glycoconjugates in the sample with the results obtained from steps (a), (b) and (d) and a computational method.    
     
     
         2 - 70 . (canceled)  
     
     
         71 . A method of analyzing a sample containing one or more glycoconjugates, which comprise one or more glycans conjugated to a non-saccharide component, comprising: 
 (a) analyzing the glycoconjugates to determine the glycosylation sites and glycosylation site occupancy,    (b) separating the glycans from the sample containing one or more glycoconjugates,    (c) analyzing the glycans to characterize the glycans, and    (d) determining the identity and quantity of all of the glycoforms of the glycoconjugates in the sample,    wherein determining the glycosylation sites and glycosylation site occupancy comprises cleaving the glycans from the non-saccharide components and labeling the non-saccharide components at their glycosylation sites of a first portion of the sample, cleaving the glycans from the non-saccharide components of a second portion of the sample, analyzing the first and second portions of the sample of glycoconjugates and comparing the results.    
     
     
         72 - 90 . (canceled)  
     
     
         91 . A method of analyzing a sample containing one or more carbohydrates, comprising: 
 (a) analyzing the carbohydrates with MALDI-MS to determine the monomer composition and relative abundance of the carbohydrates,    (b) analyzing the carbohydrates with NMR to determine the monomer composition and linkage abundance of the carbohydrates,    (c) and generating constraints from the results of (a) and (b) and solving the constraints with a computational method.    
     
     
         92 . The method of  claim 91 , wherein the carbohydrates are polysaccharides.  
     
     
         93 . The method of  claim 92 , wherein the polysaccharides are glycans.  
     
     
         94 . The method of  claim 93 , wherein the glycans are branched.  
     
     
         95 . The method of  claim 93 , wherein the glycans are conjugated to a non-saccharide component and form one or more glycoconjugates.  
     
     
         96 . The method of  claim 95 , wherein the method further comprises analyzing the non-saccharide components or one or more glycoconjugates or a combination thereof.  
     
     
         97 . The method of  claim 93 , wherein further constraints are generated from databases containing information about glycans, non-saccharide components, glycoconjugates or a combination thereof.  
     
     
         98 . The method of  claim 93 , wherein further constraints are generated from biosynthetic rules.  
     
     
         99 . The method of  claim 91 , wherein further constraints are generated by analyzing the carbohydrates with another experimental method.  
     
     
         100 . The method of  claim 99 , wherein the other experimental method is a mass spectrometric method that is not MALDI-MS, an electrophoretic method, a different NMR method, a chromatographic method or some combination thereof.  
     
     
         101 . The method of  claim 91 , wherein further constraints are generated from information about the sample origin.  
     
     
         102 . The method of  claim 91 , wherein the constraints are one or more mathematical equations and are solved by determining the solution of the one or more mathematical equations.  
     
     
         103 . The method of  claim 91 , wherein the constraints are solved with a computer program.  
     
     
         104 . The method of  claim 93 , wherein the method further comprises generating a list of all possible glycans.  
     
     
         105 . The method of  claim 93 , wherein neutral and charged glycans are analyzed separately.  
     
     
         106 . The method of  claim 91 , wherein the MALDI-MS is performed in the presence of a thymine derivative and an ion exchange resin.  
     
     
         107 . The method of  claim 93 , wherein a mixture of glycans is analyzed.  
     
     
         108 - 146 . (canceled)  
     
     
         147 . A method of analyzing a sample containing glycans, comprising: 
 analyzing a first portion of the sample, wherein the glycans have been removed,    analyzing a second portion of the sample, wherein the second portion of the sample contains intact glycoconjugates, which comprise one or more glycans conjugated to a non-saccharide component,    and analyzing a third portion of the sample, wherein the third portion of the sample contains glycans.    
     
     
         148 - 163 . (canceled)  
     
     
         164 . A method of generating a glycoconjugate library, wherein the glycoconjugate comprises one or more glycans conjugated to a non-saccharide component, comprising: 
 cleaving the non-saccharide component of the glycoconjugate in a sample and labeling the non-saccharide component fragments generated with a labeling agent in order to generate a glycoconjugate library, and    cleaving the glycans from the non-saccharide components and labeling the non-saccharide components in the sample at the glycosylation sites.    
     
     
         165 - 171 . (canceled)  
     
     
         172 . A library generated with the method of  claim 164 .  
     
     
         173 . A method of analyzing a sample containing glycoconjugates, comprising: 
 modifying the glycoconjugates in the sample, and    comparing the modified glycoconjugates with the library of  claim 164 .    
     
     
         174 - 179 . (canceled)  
     
     
         180 . A method of generating a list of properties, comprising: 
 determining one or more properties of a sample with the method of  claim 1 , and    recording a value for the one or more properties to generate a list, wherein the value of the one or more properties is recorded in a computer-generated data structure.    
     
     
         181 - 222 . (canceled)  
     
     
         223 . A method of analyzing the total glycome of a sample, comprising: 
 (a) analyzing all of the glycans of the sample, and    (b) determining a profile of the glycans of the sample, wherein the glycans of the sample are analyzed with a method, comprising:    analyzing the glycans with MALDI-MS to determine the monosaccharide composition and relative abundance of the glycans, and    analyzing the glycans with NMR to determine the monosaccharide composition and linkage abundance of the glycans.    
     
     
         224 - 295 . (canceled)  
     
     
         296 . A method of generating a glycoprofile according to the method of  claim 223 .  
     
     
         297 . A method of creating a database of glycoprofiles, comprising: 
 generating a glycoprofile of a sample according to the method of  claim 296 , and    recording one or more values corresponding to the glycoprofile in a computer-generated data structure.    
     
     
         298 . The database created according to the method of  claim 297 .  
     
     
         299 . A method of determining a glycome pattern, comprising: 
 obtaining a glycoprofile of total glycans of a sample containing glycans according to the method of  claim 223 ,    identifying features of the glycoprofile,    generating data sets based on the features of the glycoprofile,    identifying a pattern in the data sets,    and determining whether or not the pattern is associated with a known sample or diseased state.    
     
     
         300 - 310 . (canceled)  
     
     
         311 . A method of determining a glycome pattern of a sample, comprising: 
 determining the glycoprofile of the sample according to the method of  claim 223 ,    extracting one or more features of the glycoprofile,    analyzing the one or more features,    and validating the glycome pattern.    
     
     
         312 - 328 . (canceled)