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

Spontaneous conversion of aspartic acid (Asp) to isoasparticacid(isoAsp) is a ubiquitous modification that influencesthe structure and function of proteins. This modification of Asp impactsthe stability of biotherapeutics and has been linked to the developmentof neurodegenerative diseases. We explored the use of 193 nm ultravioletphotodissociation (UVPD) to distinguish Asp and isoAsp in the protonated and deprotonated peptides. The differencesin the relative abundances of several fragment ions uniquely generatedby UVPD were used to differentiate isomeric peptide standards containingAsp or isoAsp. These fragment ions result from thecleavage of bonds N-terminal to Asp/isoAsp residuesin addition to the side-chain losses from Asp/isoAsp or the losses of COOH, CO2, CO, or H(2)Ofrom y-ions. Fragmentation of Asp-containing trypticpeptides using UVPD resulted in more enhanced w/w + 1/y - 1/x ions,while isoAsp-containing peptides yielded more enhanced y - 18/y - 45/y - 46 ions. UVPD was also used to identify an isomerized peptidefrom a tryptic digest of a monoclonal antibody. Moreover, UVPD ofa protonated nontryptic peptide resulted in more enhanced y ions N- and C-terminal to isoAsp anddifferences in b/y ion ratios thatwere used to identify the isoAsp peptide.