Methods to prevent disulfide scrambling for MS-based proteomics
The present invention generally pertains to methods of preventing disulfide scrambling in non-reducing liquid chromatography-mass spectrometry analysis of a protein of interest. In particular, the present invention pertains to the addition of maleimide to a non-reducing liquid chromatography-mass spectrometry analysis of a protein to prevent disulfide scrambling.
1 . A method for performing a non-reduced peptide mapping of a protein of interest in a sample, said method comprising:
a. contacting said sample to a NEM analog to form an alkylated protein of interest, wherein the NEM analog is maleimide or N-hydroxy maleimide;
b. contacting said alkylated protein of interest to at least one digestive enzyme to form a peptide digest; and
c. analyzing said peptide digest using liquid chromatography-mass spectrometry to obtain said non-reduced peptide mapping of said protein of interest.
2 . The method of claim 1 , wherein the NEM analog is maleimide.
3 . The method of claim 1 , wherein the concentration of NEM analog used to contact said sample is about 1 mM to about 10 mM.
4 . The method of claim 1 , wherein the concentration of NEM analog used to contact said sample is about 2 mM to about 8 mM.
5 . The method of claim 1 , wherein the concentration of NEM analog used to contact said sample is about 4 mM.
6 . The method of claim 1 , wherein said NEM analog is contacted with said sample at 50° C. for 30 minutes.
7 . The method of claim 1 , wherein said protein of interest is an antibody.
8 . The method of claim 1 , further comprising contacting said sample to at least one denaturation agent.
9 . The method of claim 1 , wherein said at least one digestive enzyme is trypsin.
10 . The method of claim 1 , wherein said at least one digestive enzyme is Lys-C.
11 . The method of claim 1 , wherein said at least one digestive enzyme is Lys-C and trypsin.
12 . The method of claim 1 , wherein said digestion is conducted at a pH of about 7 to about 8.
13 . The method of claim 1 , wherein said digestion is conducted at a pH of about 7 to about 7.5.
14 . The method of claim 1 , wherein said digestion is conducted at a pH of about 5 to about 6.
15 . The method of claim 1 , wherein said digestion is conducted at a pH of about 5.3 to about 7.
16 . A method for characterizing a protein of interest in a sample, said method comprising:
a. contacting said sample to a NEM analog to form an alkylated protein of interest, wherein the NEM analog is maleimide or N-hydroxy maleimide;
b. contacting said alkylated protein of interest to at least one digestive enzyme to form a peptide digest; and
c. analyzing said peptide digest using liquid chromatography-mass spectrometry to obtain a non-reduced peptide mapping to characterize said protein of interest.
17 . The method of claim 16 , wherein the NEM analog is maleimide.
18 . The method of claim 17 , wherein the concentration of maleimide used to contact said sample is about 2 mM to about 8 mM.
19 . The method of claim 18 , wherein the concentration of maleimide used to contact said sample is about 4 mM.
20 . The method of claim 16 , further comprising contacting said sample to at least one denaturation agent.