Methods for characterizing a protein of interest
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsKLEINBERG Andrew, MAO Yuan, LI Ning
Life of this patent
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Abstract
Methods can be conducted to prevent disulfide scrambling in liquid chromatography-mass spectrometry analysis of a protein of interest. In particular, methods can be conducted by employing a NEM analog, such as maleimide, and a heavy isotope counterpart of the NEM analog, such as a heavy isotope substituted maleimide, to alkylate a protein of interest and to alkylate a peptide digest of the protein of interest under conditions that prevent disulfide scrambling and subjecting the samples to a liquid chromatography-mass spectrometry analysis.
Claims20
A method for site-specific free thiol quantitation in a first sample including a protein of interest, the method comprising:
- alkylating the protein of interest in the first sample with an alkylating agent to form a second sample with an alkylated protein of interest, wherein the alkylating agent is either:
- (i) a NEM analog, or (ii) a heavy isotope substituted form of the NEM analog;
- digesting the alkylated protein of interest with at least one digestive enzyme to form a peptide digest;
- alkylating the peptide digest with a differential alkylating agent to form a third sample, wherein the differential alkylating agent is either (i) the NEM analog, or (ii) the heavy isotope substituted form of the NEM analog;
- and
- subjecting the third sample to analysis using liquid chromatography-mass spectrometry to quantify the site-specific free thiol in the first sample.
A method for characterizing a protein of interest in a first sample, the method comprising:
- alkylating the protein of interest in the first sample with an alkylating agent to form a second sample with an alkylated protein of interest, wherein the alkylating agent is either:
- (i) a NEM analog, or (ii) a heavy isotope substituted form of the NEM analog;
- digesting the alkylated protein of interest with at least one digestive enzyme to form a peptide digest;
- alkylating the peptide digest with a differential alkylating agent to form a third sample, wherein the differential alkylating agent is either:
- (i) the NEM analog, or (ii) the heavy isotope substituted form of the NEM analog;
- and
- subjecting the third sample to analysis using liquid chromatography-mass spectrometry to characterize the protein of interest in the first sample.
A method for characterizing a protein of interest in a first sample, the method comprising:
- forming an alkylated product by either:
- alkylating the protein of interest with a carbon 13 substituted NEM analog to form a 13C-NEM analog alkylated protein of interest, or alkylating a peptide digest of the protein of interest with a carbon 13 substituted NEM analog to form a 13C-NEM analog alkylated peptide digest.
Dependencies are inferred from the claim text.