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. Improved methods, therefore, for the study of glycosylation patterns on cells, tissue and body fluids are also provided. Information regarding 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 on protein function. Such methods are also provided. Methods are also provided to assess protein production processes, to assess the purity of proteins produced, and to select proteins with the desired glycosylation.

1 . A method of analyzing a sample containing glycoconjugates, comprising: 
 (a) separating the glycans from the sample containing the glycoconjugates,    (b) determining the glycosylation sites and glycosylation site occupancy of the glycoconjugates,    (c) analyzing the glycans to characterize the glycans, and    (d) determining the glycoforms of the glycoconjugates in the sample with the results obtained from steps (b) and (c) with a computational method.    
   
   
       2 .- 10 . (canceled)  
   
   
       11 . The method of  claim 1 , wherein the glycoconjugates comprise a peptide and wherein the step of determining the glycosylation sites and glycosylation site occupancy, comprises 
 cleaving the backbone of the glycoconjugates,    cleaving and labeling with a first label the glycoconjugates of a first portion of the sample at their glycosylation sites,    cleaving the glycoconjugates of a second portion of the sample at their glycosylation sites,    analyzing the first and second portions of the sample of glycoconjugates, and    quantifying the results of the analysis step.    
   
   
       12 .- 31 . (canceled)  
   
   
       32 . A method of analyzing a sample containing glycoconjugates, comprising: 
 separating the glycans from the sample containing the glycoconjugates,    determining the glycosylation sites and glycosylation site occupancy of the glycoconjugates, and    analyzing the glycans to characterize the glycans,    wherein determining the glycosylation sites and glycosylation site occupancy comprises cleaving and labeling with a first label the glycoconjugates at their glycosylation sites of a first portion of the sample, cleaving the glycoconjugates at their glycosylation sites of a second portion of the sample, analyzing the first and second portions of the sample of glycoconjugates, and quantifying the results.    
   
   
       33 .- 46 . (canceled)  
   
   
       47 . A method of determining the glycosylation site occupancy of glycoconjugates in a sample, comprising: 
 cleaving and labeling with a first label the glycoconjugates at their glycosylation sites of a first portion of the sample, cleaving the glycoconjugates at their glycosylation sites of a second portion of the sample, analyzing the first and second portions of the sample of glycoconjugates, and quantifying the results.    
   
   
       48 .- 54 . (canceled)  
   
   
       55 . A method of analyzing a sample containing glycans, comprising: 
 separating neutral from charged glycans, and    analyzing the neutral and charged glycans separately to characterize the glycan.    
   
   
       56 . (canceled)  
   
   
       57 . A method of analyzing a glycan, comprising: 
 analyzing the glycan in the presence of Nafion and ATT.    
   
   
       58 . The method of  claim 1 , wherein the method is a method of analyzing the purity of a sample containing glycans.  
   
   
       59 . The method of  claim 1 , wherein the method is a method of analyzing the glycans of a sample of one or more cells, a tissue or body fluid from a subject.  
   
   
       60 . A high-throughput method of  claim 1 , wherein more than one sample is analyzed.  
   
   
       61 .- 62 . (canceled)  
   
   
       63 . A method of generating a glycopeptide library, comprising: 
 cleaving the backbone of the glycopeptides in a sample and labeling the glycopeptide fragments generated with a labeling agent, and    cleaving the glycans in the sample.    
   
   
       64 .- 68 . (canceled)  
   
   
       69 . A library of glycopeptides generated with the method of  claim 63 .  
   
   
       70 . A method of analyzing a sample of glycopeptides, comprising: 
 characterizing the glycopeptides in the sample, and    comparing the characterized glycopeptides with the library of glycopeptides of  claim 69 .    
   
   
       71 . A method of generating a list of glycoconjugate properties, comprising: 
 measuring two or more properties of the glycoconjugate, and    recording a value for the two or more properties of the glycoconjugate to generate a list, wherein the value of the two or more properties is recorded in a computer-generated data structure.    
   
   
       72 .- 77 . (canceled)  
   
   
       78 . A database, tangibly embodied in a computer-readable medium, for storing information descriptive of one or more glycoconjugates, the database comprising: 
 one or more data units corresponding to the one or more glycoconjugates, each of the data units including an identifier that includes two or more fields, each field for storing a value corresponding to one or more properties of the glycoconjugates.    
   
   
       79 . A method of analyzing the total glycome of a sample of body fluid, comprising: 
 (a) analyzing the glycans of the sample, and    (b) determining a profile of the glycans of the sample.    
   
   
       80 . The method of  claim 79 , wherein the method further comprises performing a pattern analysis on the results from (a) using a computational method.  
   
   
       81 .- 85 . (canceled)  
   
   
       86 . A method of analyzing the total glycome of a sample of body fluid, comprising: 
 (a) analyzing the glycans of a sample of body fluid, and    (b) comparing the results from (a) with a known pattern.    
   
   
       87 .- 100 . (canceled)  
   
   
       101 . A method of determining the purity of a sample, comprising: 
 (a) analyzing the total glycans of the sample,    (b) identifying the glycan pattern of the sample, and    (b) comparing the pattern with a known pattern of a sample of predetermined purity to assess the purity of the sample.    
   
   
       102 . A method of generating the complete glycan profile of a body fluid, comprising: 
 (a) analyzing the glycans in the sample, and    (b) identifying the complete glycan profile of the sample.    
   
   
       103 .- 104 . (canceled)  
   
   
       105 . A method of analyzing the total glycome of a sample, comprising: 
 determining the glycosylation site and glycosylation site occupancy of all glycoconjugates in the sample,    characterizing components of the glycoconjugates and all glycans of the glycome in the sample,    and matching specific glycans to glycoconjugates with a computational method.    
   
   
       106 . A method of analyzing a sample of glycoconjugates, comprising: 
 analyzing the glycans of the sample with an analytical method, and    determining the glycoforms of the sample with a computational method.    
   
   
       107 . (canceled)