Maximizing hydrophobic peptide recovery using a mass spectrometry compatible surfactant
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.
1 . A method 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;
wherein the protein of interest is an antibody having a peptide from a complementarity-determining region (CDR) of the antibody, and wherein the sample with the DDM exhibits an increased signal for the peptide from the CDR relative to a signal for the peptide from the CDR in a sample without DDM.
2 . The method of claim 1 , wherein said peptide mapping analysis is reduced peptide mapping analysis.
3 . The method of claim 1 , wherein said protein of interest is a bispecific antibody, a monoclonal antibody, or an antibody-drug conjugate.
4 . The method of claim 1 , wherein said conditions suitable for denaturing comprise heating said sample, optionally wherein said heating is conducted at about 80° C. for about 10 minutes.
5 . The method of claim 1 , further comprising subjecting said sample to conditions suitable for reduction.
6 . The method of claim 5 , wherein said conditions suitable for reduction comprise contacting said sample to Tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl), optionally wherein a concentration of said TCEP-HCl is about 5 mM.
7 . The method of claim 1 , wherein said at least one alkylating agent comprises iodoacetamide.
8 . The method of claim 1 , wherein a final concentration of DDM in said mixture is about 0.1%.
9 . The method of claim 1 , wherein said at least one digestive enzyme comprises trypsin, optionally wherein said trypsin is present at an enzyme to substrate ratio of about 1:10 (w/w).
10 . The method of claim 1 , wherein said liquid chromatography-mass spectrometry comprises reversed-phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed-mode chromatography, or a combination thereof.
11 . The method of claim 1 , wherein said liquid chromatography-mass spectrometry comprises a mass spectrometer coupled to liquid chromatography, and said mass spectrometer is an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer.
12 . The method of claim 1 , wherein said liquid chromatography-mass spectrometry analysis comprises parallel reaction monitoring analysis.
13 . The method of claim 1 , wherein the peptide mapping analysis further comprises obtaining an ultraviolet signal of the peptide from the CDR by an ultraviolet spectrometer coupled to the liquid chromatography-mass spectrometry.
14 . The method of claim 13 , wherein the sample with the DDM exhibits an increased ultraviolet signal for the peptide from the CDR relative to an ultraviolet signal for the peptide from the CDR in a sample without DDM.
15 . The method of claim 13 , wherein the sample with the DDM exhibits the ultraviolet signal with a reduced relative standard deviation (RSD) for the peptide from the CDR relative to a ultraviolet signal of the peptide from the CDR in a sample without DDM.
16 . The method of claim 1 , wherein the peptide digest is dried and reconstituted with a solution including N-dodecyl-β-D-maltoside prior to subjecting said peptide digest to the liquid chromatography-mass spectrometry for the peptide mapping analysis.
17 . The method of claim 1 , wherein a final concentration of DDM in said mixture is from about 0.01% to about 0.1%.
18 . The method of claim 1 , wherein said peptide mapping analysis is a non-reduced peptide mapping analysis.