title: Characterization of succinimide stability during trypsin digestion for LC-MS analysis.
abstract: LC-MS peptide mapping is the most commonly used method to analyze protein modifications. The proteins are generally digested using trypsin at a slightly basic pH at 37 °C from several hours to overnight. Assay-induced artifacts can be generated during this procedure, potentially causing false-positive or false-negative results for a given modification. Unfortunately, for the analysis of succinimide, both false-negative and false-positive results can be generated within the same procedure. This study evaluates the stability of succinimide during the peptide mapping procedure and has demonstrated that up to 13% of pre-existing succinimide was lost during a 4 h trypsin digestion at pH 5.0 which was previously determined to be optimal for the detection of succinimide. The same procedure was able to simultaneously generate approximately 3% succinimide. Using the optimized procedure, it was also found that two aspartate residues that are followed by glycine residues in the conserved Fc region of a recombinant monoclonal antibody were not prone to isomerization. On the other hand, an aspartate residue followed by a glycine in the heavy chain variable domain was highly susceptible to isomerization. Interestingly, the antibody containing the succinimide eluted from an SEC column after the monomer peak.
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT 06510, United States.
authors.forename: Christine
authors.initials: C
authors.lastname: Nowak
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT 06510, United States.
authors.forename: Gomathinayagam
authors.initials: G
authors.lastname: Ponniah
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT 06510, United States.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Neill
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT 06510, United States. Electronic address: LiuH@alxn.com.
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Liu
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D010449:Peptide Mapping; D013388:Succinimides / Q000652:urine*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D013388:Succinimides; D001224:Aspartic Acid; D009113:Muramidase; D014357:Trypsin
keywords: Mass spectrometry; Recombinant monoclonal antibody; Succinimide; Trypsin digestion