| Autor: |
Bashyal A; Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States., Hui JO; Amgen Research, Molecular Analytics, Amgen Inc., Thousand Oaks, California 91320, United States., Flick T; Process Development, Attribute Sciences, Amgen Inc., Thousand Oaks, California 91320, United States., Dykstra AB; Process Development, Attribute Sciences, Amgen Inc., Thousand Oaks, California 91320, United States., Zhang Q; Process Development, Attribute Sciences, Amgen Inc., Thousand Oaks, California 91320, United States., Campuzano IDG; Amgen Research, Molecular Analytics, Amgen Inc., Thousand Oaks, California 91320, United States., Brodbelt JS; Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States. |
| Abstrakt: |
Spontaneous conversion of aspartic acid (Asp) to isoaspartic acid ( iso Asp) is a ubiquitous modification that influences the structure and function of proteins. This modification of Asp impacts the stability of biotherapeutics and has been linked to the development of neurodegenerative diseases. We explored the use of 193 nm ultraviolet photodissociation (UVPD) to distinguish Asp and iso Asp in the protonated and deprotonated peptides. The differences in the relative abundances of several fragment ions uniquely generated by UVPD were used to differentiate isomeric peptide standards containing Asp or iso Asp. These fragment ions result from the cleavage of bonds N-terminal to Asp/ iso Asp residues in addition to the side-chain losses from Asp/ iso Asp or the losses of COOH, CO 2 , CO, or H 2 O from y -ions. Fragmentation of Asp-containing tryptic peptides using UVPD resulted in more enhanced w / w + 1/ y - 1/ x ions, while iso Asp-containing peptides yielded more enhanced y - 18/ y - 45/ y - 46 ions. UVPD was also used to identify an isomerized peptide from a tryptic digest of a monoclonal antibody. Moreover, UVPD of a protonated nontryptic peptide resulted in more enhanced y ions N- and C-terminal to iso Asp and differences in b / y ion ratios that were used to identify the iso Asp peptide. |
