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isoAsp-Quest: workflow development for isoAsp identification using database searches
Hiroaki Sakaue1, Atsushi Kuno1
1Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.
Journal of Biochemistry
|October 23, 2024
Summary
A new method, isoAsp-Quest, identifies aspartyl isomerization in proteins. This technique uses enzymatic conversion and mass spectrometry to detect subtle changes, aiding protein quality control and biomarker discovery.
Area of Science:
- Biochemistry
- Proteomics
- Analytical Chemistry
Background:
- Aspartyl residue isomerization is a common post-translational modification in vivo.
- Standard mass spectrometry struggles to detect Asp isomerization due to unchanged molecular weight.
Purpose of the Study:
- To develop a novel mass spectrometry-based method for identifying aspartyl isomerization (isoAsp).
- To establish a straightforward and rapid workflow for analyzing isoAsp modifications.
Main Methods:
- Developed isoAsp-Quest, a database search-oriented method.
- Utilized protein L-isoaspartyl-O-methyltransferase (PIMT) for enzymatic conversion to 18O-labelled Lα-Asp.
- Employed AspN degradation to remove endogenous Lα-Asp before PIMT reaction for enhanced detection.
Main Results:
- Successfully identified several Asp isomerization sites in bovine lens α-crystallin.
- Results were consistent with findings in human αA-crystallin, validating the method's effectiveness.
- Demonstrated the utility of isoAsp-Quest for analyzing Lβ-Asp in biological samples.
Conclusions:
- The isoAsp-Quest strategy provides a robust method for analyzing aspartyl isomerization.
- This approach is valuable for protein product quality control and biomarker discovery.
- The method enables straightforward and rapid identification of isoAsp modifications.
