title: A conventional procedure to reduce Asn deamidation artifacts during trypsin peptide mapping.
abstract: Asn deamidation is a common post-translational modification of proteins with significant biological consequences. Asn deamidation can cause changes in structure, stability and function of proteins. LC-MS peptide mapping is the most widely used method to detect and quantify Asn deamidation. However, a significant amount of deamidation can occur during sample preparation for peptide mapping, making it challenging to accurately determine the original level of deamidation. Although several protocols to reduce procedure-induced deamidation have been reported, they either require special procedural steps or are not optimal for maintaining trypsin activity. In the current study, several commonly used buffers that are optimal for trypsin activity were evaluated. The results demonstrated that much lower levels of Asn deamidation artifacts were observed when Tris buffer was used, especially at lower concentrations. The addition of 10% acetonitrile further reduced the levels of Asn deamidation artifacts. The utility of the optimized procedure was demonstrated by the digestion of a recombinant monoclonal antibody. The proposed procedure can be readily applied to any laboratory settings as it does not require any special reagents or procedures.
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, CT 06410, USA.
authors.forename: Yekaterina
authors.initials: Y
authors.lastname: Kori
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, CT 06410, USA.
authors.forename: Rekha
authors.initials: R
authors.lastname: Patel
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, CT 06410, USA.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Neill
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, CT 06410, USA. Electronic address: liuh@alxn.com.
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Liu
mesh_terms: D000478:Alkylation; D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000378:metabolism; D016477:Artifacts; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
chemical_list: D000577:Amides; D000911:Antibodies, Monoclonal; D002021:Buffers; D010455:Peptides; D014357:Trypsin
keywords: Deamidation; Isoaspartate; Mass spectrometry; Monoclonal antibody; Peptide mapping