Methods for quantitative proteome analysis of glycoproteins

Abstract

The invention provides a method for identifying and quantifying polyglycopeptides in a sample. The method can include the steps of immobilizing glycopolypeptides to a solid support; cleaving the immobilized glycopolypeptides, thereby releasing non-glycosylated peptides and retaining immobilized glycopeptides; releasing the glycopeptides from the solid support; and analyzing the released glycopeptides. The method can further include the step of identifying one or more glycopeptides, for example, using mass spectrometry.

1 - 42 . (canceled)  
     
     
         43 . A method for reducing peptide complexity in a sample containing glycopolypeptides, comprising: 
 (a) immobilizing to a solid support a derivatized carbohydrate group of a glycopolypeptide in the sample;    (b) cleaving said immobilized glycopolypeptides, thereby releasing non-glycosylated peptide fragments and retaining immobilized glycopeptide fragments;    (c) labeling said immobilized glycopeptide fragments with a tag; and    (d) releasing said glycopeptide fragments from said solid support, thereby generating released glycopeptide fragments;    thereby reducing peptide complexity in the sample.    
     
     
         44 . The method of  claim 43 , wherein said solid support comprises a hydrazide moiety.  
     
     
         45 . The method of  claim 43 , wherein said glycopeptides are released from said solid support using a glycosidase.  
     
     
         46 . The method of  claim 45 , wherein said glycosidase is an N-glycosidase or an O-glycosidase.  
     
     
         47 . The method of  claim 46 , wherein said glycopeptides are released from said solid support using sequential addition of N-glycosidase and O-glycosidase.  
     
     
         48 . The method of  claim 43 , wherein said glycopeptides are released from said solid support using chemical cleavage.  
     
     
         49 . The method of  claim 43 , wherein said glycopolypeptides are oxidized with periodate.  
     
     
         50 . The method of  claim 43 , wherein said glycopolypeptides are cleaved with trypsin.  
     
     
         51 . The method of  claim 43 , wherein the tag is an isotope tag.  
     
     
         52 . The method of  claim 43 , wherein said released non-glycosylated peptides are isotopically labeled.  
     
     
         53 . The method of  claim 43 , wherein said sample is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, urine, and lung lavage.  
     
     
         54 . A method for identifying a profile of markers, comprising: 
 (a) immobilizing to a first solid support a derivatized carbohydrate group of a glycopolypeptide in a test sample obtained from an individual having a disease;    (b) immobilizing to a second solid support a derivatized carbohydrate group of a glycopolypeptide in a control sample;    (c) cleaving the immobilized glycopolypeptides on the first and second supports, thereby releasing non-glycosylated peptides and retaining immobilized glycopeptides;    (d) labeling the immobilized glycopeptides on the first and second supports with a tag;    (e) releasing the glycopeptides from the solid supports;    (f) analyzing the released glycopeptides; and    (g) identifying one or more glycosylated polypeptides having differential glycosylation between the test sample and the control sample;    thereby identifying a profile of markers.    
     
     
         55 . The method of  claim 54  wherein the differential glycosylation comprises the presence or absence of a glycopolypeptide in the test sample compared to the control sample.  
     
     
         56 . The method of  claim 54  wherein the differential glycosylation comprises quantitative differential expression in one or more glycopolypeptides in the test sample compared to the control sample.  
     
     
         57 . The method of  claim 54  wherein step (d) comprises labeling the immobilized glycopeptides on the first and second supports with differential tags on the respective supports.  
     
     
         58 . The method of  claim 54  wherein the test and control samples are run in parallel and analyzed separately.  
     
     
         59 . The method of  claim 54 , wherein said first and second solid support comprises a hydrazide moiety.  
     
     
         60 . The method of  claim 54 , wherein said glycopeptides are released from said first and second solid support using a glycosidase.  
     
     
         61 . The method of  claim 60 , wherein said glycosidase is an N-glycosidase or an O-glycosidase.  
     
     
         62 . The method of  claim 61 , wherein said glycopeptides are released from said first and second solid support using sequential addition of N-glycosidase and O-glycosidase.  
     
     
         63 . The method of  claim 54 , wherein said glycopeptides are released from said first and second solid support using chemical cleavage.  
     
     
         64 . The method of  claim 54 , wherein said glycopolypeptides are oxidized with periodate.  
     
     
         65 . The method of  claim 54 , wherein said glycopolypeptides are cleaved with trypsin.  
     
     
         66 . The method of  claim 54 , wherein said released non-glycosylated peptides are isotopically labeled.  
     
     
         67 . The method of  claim 54  wherein the sample is selected from the group consisting of blood, serum, plasma, tissue biopsy, cerebrospinal fluid, urine, and lung lavage.