Maximizing hydrophobic peptide recovery using a mass spectrometry compatible surfactant
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsSong Nie, Reid O'Brien Johnson, Tyler Greer, Xiaojing Zheng
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Abstract
The present invention generally pertains to methods of characterizing peptides or proteins of interest. In particular, the present invention pertains to the use of the nonionic surfactant DDM to improve recovery of hydrophobic peptides in pharmacokinetic, reduced peptide mapping, or other biotherapeutic protein analyses.
Claims37
A method for preparing a peptide standard for a pharmacokinetic study for liquid chromatography-mass spectrometry analysis, comprising:
- (a) contacting a peptide standard for a pharmacokinetic study to N-dodecyl-β-D-maltoside (DDM) to form a mixture;
- and
- (b) subjecting said mixture to liquid chromatography-mass spectrometry analysis.
A method for selecting a target peptide for a pharmacokinetic study, comprising:
- (a) obtaining at least two candidate peptide standards having the amino acid sequence of at least two candidate target peptides for a pharmacokinetic study;
- (b) contacting each of said at least two candidate peptide standards to N-dodecyl-β-D-maltoside (DDM) to form at least two mixtures;
- (c) subjecting each of said at least two mixtures to liquid chromatography-mass spectrometry analysis to produce mass spectra for each of said at least two candidate peptide standards;
- (d) comparing each of said mass spectra to mass spectra produced by liquid chromatography-mass spectrometry analysis of a control mixture to produce a ratio of average peak area for each of said at least two candidate peptide standards;
- (e) comparing said ratio of average peak area between each of said at least two candidate peptide standards;
- and
- (f) using the comparison of (e) to select a target peptide for a pharmacokinetic study,
- wherein said control mixture includes the candidate peptide standard and does not include DDM.
A method for preparing a protein of interest for peptide mapping analysis, comprising:
- (a) subjecting a sample including a protein of interest to conditions suitable for denaturing to form a denatured sample;
- (b) contacting said denatured sample to at least one alkylating agent under conditions suitable for alkylation to form an alkylated sample;
- (c) contacting said alkylated sample to N-dodecyl-β-D-maltoside (DDM) to form a mixture;
- (d) contacting said mixture to at least one digestive enzyme under conditions suitable for digestion to form a peptide digest;
- and
- (e) subjecting said peptide digest to liquid chromatography-mass spectrometry for a peptide mapping analysis.
A method for preparing a protein sample of interest for liquid chromatography-mass spectrometry analysis, comprising:
- (a) drying a protein sample of interest using a speed vacuum to form a dried sample;
- (b) dissolving said dried sample using a solution including N-dodecyl-β-D-maltoside (DDM) to form a reconstituted sample;
- and
- (c) subjecting said reconstituted sample to liquid chromatography-mass spectrometry analysis.
Dependencies are inferred from the claim text.