Analysis of isoaspartic Acid by selective proteolysis with Asp-N and electron transfer dissociation mass spectrometry.
A ubiquitous yet underappreciated protein post-translational modification, isoaspartic acid (isoAsp, isoD, or beta-Asp), generated via the deamidation of asparagine or isomerization of aspartic acid in proteins, plays a diverse and crucial role in aging, as well as autoimmune, cancer, neurodegeneration, and other diseases. In addition, formation of isoAsp is a major concern in protein pharmaceuticals, as it may lead to aggregation or activity loss. The scope and significance of isoAsp have, up to now, not been fully explored, as an unbiased screening of isoAsp at low abundance remains challenging. This difficulty is due to the subtle difference in the physicochemical properties between isoAsp and Asp, e.g., identical mass. In contrast, endoprotease Asp-N (EC 3.4.24.33) selectively cleaves aspartyl peptides but not the isoaspartyl counterparts. As a consequence, isoaspartyl peptides can be differentiated from those containing Asp and also enriched by Asp-N digestion. Subsequently, the existence and site of isoaspartate can be confirmed by electron transfer dissociation (ETD) mass spectrometry. As little as 0.5% of isoAsp was detected in synthetic beta-amyloid and cytochrome c peptides, even though both were initially assumed to be free of isoAsp. Taken together, our approach should expedite the unbiased discovery of isoAsp.
Ni
Wenqin
W
Barnett Institute of Chemical and Biological Analysis and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Dai
Shujia
S
Karger
Barry L
BL
Zhou
Zhaohui Sunny
ZS
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D045304:Cytochromes c / Q000737:chemistry; D004579:Electron Transport; D026581:Isoaspartic Acid / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data
D000682:Amyloid; D026581:Isoaspartic Acid; D045304:Cytochromes c; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N