Differentiation of Aspartic and Isoaspartic Acid Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Spontaneous conversion of aspartic acid (Asp) to isoaspartic acid (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 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 Asp. These fragment ions result from the cleavage of bonds N-terminal to Asp/Asp residues in addition to the side-chain losses from Asp/Asp or the losses of COOH, CO, CO, or HO from -ions. Fragmentation of Asp-containing tryptic peptides using UVPD resulted in more enhanced / + 1/ - 1/ ions, while Asp-containing peptides yielded more enhanced - 18/ - 45/ - 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 ions N- and C-terminal to Asp and differences in / ion ratios that were used to identify the Asp peptide.