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

Abstract

Spontaneous conversion of aspartic acid (Asp) to isoaspartic acid (isoAsp) 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 isoAsp 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 isoAsp. These fragment ions result from the cleavage of bonds N-terminal to Asp/isoAsp residues in addition to the side-chain losses from Asp/isoAsp or the losses of COOH, CO2, CO, or H2O from y-ions. Fragmentation of Asp-containing tryptic peptides 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 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 isoAsp and differences in b/y ion ratios that were used to identify the isoAsp peptide.

Deuterium Labeling of Isoaspartic and Isoglutamic Acids for Mass Spectrometry Analysis

“…These strategies are based on (1) liquid chromatography (LC) or ion mobility mass spectrometry (IM-MS) separation, (2) diagnostic fragment ion analysis, or (3) protein l -isoaspartyl methyltransferase (PIMT)-mediated labeling. The first strategy involves the separation of isoAsp-containing peptides from their unmodified counterparts, either through LC or IM-MS techniques. − The second strategy monitors the diagnostic fragment ions unique to isoAsp produced by electron-capture/-transfer dissociation, − photodissociation, or matrix-assisted laser desorption/ionization time-of-flight/time-of-flight with collision-induced dissociation. , The third strategy involves using PIMT to label isoAsp residues. PIMT methylates the β-carboxyl group of isoAsp and facilitates the formation of succinimide, which can react with a nucleophile, such as H 2 18 O, to generate labeled isoAsp residues. − The first approach falls short in achieving the required chemical specificity to differentiate between those of isoAsp and Asp.…”

“…The first strategy involves the separation of isoAsp-containing peptides from their unmodified counterparts, either through LC or IM-MS techniques. 7 − 11 The second strategy monitors the diagnostic fragment ions unique to isoAsp produced by electron-capture/-transfer dissociation, 12 − 14 photodissociation, 15 or matrix-assisted laser desorption/ionization time-of-flight/time-of-flight with collision-induced dissociation. 16 , 17 The third strategy involves using PIMT to label isoAsp residues.…”

Deuterium Labeling of Isoaspartic and Isoglutamic Acids for Mass Spectrometry Analysis

“…The first strategy involves the separation of isoAspcontaining peptides from their unmodified counterparts, either through liquid chromatography or ion mobility mass spectrometry (IM-MS) techniques. [7][8][9][10][11] The second strategy monitors the diagnostic fragment ions unique to isoAsp produced by electron capture/transfer dissociation (ECD/ETD), [12][13][14] photodissociation (PD), 15 or matrix-assisted laser desorption/ionization time-offlight/time-of-flight (MALDI-TOF/TOF) with collision-induced dissociation (CID). 16,17 The third strategy involves using PIMT to label isoAsp residues.…”