Methods of glycoprotein analysis

Abstract

Characterization and production of protein preparations, e.g., therapeutic glycoprotein preparations, are described.

1 .- 22 . (canceled) 
     
     
         23 . A method of characterizing higher order structure of a protein, comprising:
 obtaining a first sample comprising a labeled protein in a first state, wherein the first state comprises a first level of label;   exposing a second sample of the protein to a stressor to obtain a labeled protein in a second state, wherein the stressor comprises a second level of label, wherein the second level of label alters a higher-order structure of the protein;   using mass spectrometry (MS) to obtain a first MS signal of the labeled protein in the first state and a second MS signal the labeled protein in the second state; and   comparing the first MS signal with the second MS signal to characterize a change in the higher order structure of the protein by the stressor.   
     
     
         24 . The method of  claim 23 , wherein using mass spectrometry comprises digesting the labeled protein to produce a plurality of labeled peptides. 
     
     
         25 . The method of  claim 23 , wherein the first MS signal and the second MS signal each comprise a plurality of signals from an MS spectrum of the protein. 
     
     
         26 . The method of  claim 23 , wherein the label is an isobaric label. 
     
     
         27 . The method of  claim 26 , wherein the isobaric label comprises a TMT label, iTRAQ label, or ICAT label. 
     
     
         28 . The method of  claim 26 , wherein the isobaric label is a TMT label. 
     
     
         29 . The method of  claim 23 , wherein the first state is a native state and the second state is a non-native state. 
     
     
         30 . The method of  claim 23 , wherein the protein is glycosylated. 
     
     
         31 . The method of  claim 23 , wherein the protein is an Fc fusion protein or an antibody. 
     
     
         32 . The method of  claim 23 , wherein the second level of label is higher than the first level of label. 
     
     
         33 . The method of  claim 26 , wherein the label of the first sample of the protein and the label of the second sample of the protein have the same chemical structure but different mass. 
     
     
         34 . The method of  claim 23 , further comprising:
 exposing a third sample of the protein to a third level of label that alters a higher-order structure of the protein to obtain a labeled protein in a third state,   using mass spectrometry to obtain a third MS signal of the labeled protein in the third state; and   comparing the third MS signal with the second MS signal to characterize a change in the higher order structure of the protein.   
     
     
         35 . The method of  claim 34 , wherein using mass spectrometry comprises digesting the labeled protein to produce a plurality of labeled peptides. 
     
     
         36 . The method of  claim 35 , wherein the label is an isobaric label. 
     
     
         37 . The method of  claim 36 , wherein the isobaric label comprises a TMT label, iTRAQ label, or ICAT label. 
     
     
         38 . The method of  claim 36 , wherein the third level of label is higher than the second level of label. 
     
     
         39 . The method of  claim 38 , wherein the label of the second sample of the protein and the label of the third sample of the protein have the same chemical structure but different mass. 
     
     
         40 . The method of  claim 38 , wherein the protein is an Fc fusion protein or an antibody. 
     
     
         41 . The method of  claim 40 , wherein the protein is glycosylated.