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Localization and Quantitation of Free Sulfhydryl in Recombinant Monoclonal Antibodies by Differential Labeling with 12C and 13C Iodoacetic Acid and LC−MS Analysis
- Source :
- Analytical Chemistry. 81:8101-8108
- Publication Year :
- 2009
- Publisher :
- American Chemical Society (ACS), 2009.
-
Abstract
- A low percentage of free sulfhydryl is a common feature of recombinant monoclonal antibodies although, in theory, all cysteine residues should be involved in disulfide bonds. A differential alkylation method was developed to determine the percentage of free sulfhydryl at each cysteine residue of four recombinant monoclonal antibodies. Free sulfhydryl was first alkylated with 12C iodoacetic acid. Free sulfhydryl, resulting from the reduction of disulfide bonds, was then alkylated with 13C iodoacetic acid. Cysteine containing peptides that were modified by 13C iodoacetic acid showed a molecular weight that was 2 Da higher than the same peptide that was modified by 12C iodoacetic acid. Peptides, containing the same cysteine residues that were modified with both alkylation reagents, coeluted on reversed-phase chromatography. Analysis by mass spectrometry resulted in two partially overlapped m/z series for each cysteine containing peptide, corresponding to modification by iodoacetic acid with 12C or 13C. The percentage of free sulfhydryl was then calculated using the two m/z series at each cysteine site. A low percentage of free sulfhydryl was detected at every cysteine residue in the four antibodies studied. Although different antibodies contained different levels of free sulfhydryl, similar distribution of free sulfhydryl in the domain structures was observed in the four antibodies.
- Subjects :
- Iodoacetic acid
medicine.drug_class
Molecular Sequence Data
Peptide
Alkylation
Monoclonal antibody
Mass Spectrometry
Analytical Chemistry
law.invention
Residue (chemistry)
chemistry.chemical_compound
law
medicine
Amino Acid Sequence
Carbon Radioisotopes
Cysteine
Sulfhydryl Compounds
Chromatography, High Pressure Liquid
chemistry.chemical_classification
Carbon Isotopes
Chromatography
Molecular mass
Chemistry
Antibodies, Monoclonal
Recombinant Proteins
Iodoacetic Acid
Biochemistry
Recombinant DNA
Subjects
Details
- ISSN :
- 15206882 and 00032700
- Volume :
- 81
- Database :
- OpenAIRE
- Journal :
- Analytical Chemistry
- Accession number :
- edsair.doi.dedup.....0cd07ebd49a45b7f35e92ffc33b2eec4