Identification of Asp isomerization in proteins by ¹⁸O labeling and tandem mass spectrometry.
Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H₂¹⁸O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the ¹⁶O-containing counterparts, due to ¹⁸O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without ¹⁸O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.
Protein Analytical Chemistry, Genentech Inc, South San Francisco, CA, USA. zhang.jennifer@gene.com
Jennifer
J
Zhang
Viswanatham
V
Katta
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006868:Hydrolysis; D026581:Isoaspartic Acid / Q000737:chemistry* / Q000302:isolation & purification; D010104:Oxygen Radioisotopes / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry*; D055550:Protein Stability; D059748:Proteolysis; D013194:Staining and Labeling; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014867:Water / Q000737:chemistry
D026581:Isoaspartic Acid; D010104:Oxygen Radioisotopes; D010455:Peptides; D013388:Succinimides; D014867:Water; C032620:succinimide; D001224:Aspartic Acid