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LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics
Published on: April 9, 2017
Top-down study of β2-microglobulin deamidation
Xiaojuan Li1, Xiang Yu, Catherine E Costello
1Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, United States.
Analytical Chemistry
|July 4, 2012
Summary
Identifying isoAspartic acid (isoAsp) in intact proteins is challenging. A new MS(3) method combining CAD-ECD successfully detected isoAsp at all sites in beta2-microglobulin, enabling quantitative analysis.
Area of Science:
- Mass spectrometry
- Proteomics
- Biochemistry
Background:
- Distinguishing aspartic acid (Asp) and isoAspartic acid (isoAsp) isomers from Asn deamidation is crucial for protein analysis.
- While Electron Capture Dissociation (ECD) excels at peptide-level isomer differentiation, intact protein analysis remains difficult.
- Beta2-microglobulin (β(2)M) serves as a model system for studying protein deamidation.
Purpose of the Study:
- To develop and validate a top-down mass spectrometry approach for identifying isoAsp formation in intact proteins.
- To overcome limitations of standard ECD for isoAsp detection in large molecules.
- To enable quantitative analysis of isoAsp formation in aged proteins.
Main Methods:
- Top-down mass spectrometry was employed using beta2-microglobulin (β(2)M) as a model.
- Initial top-down ECD analysis was performed on intact, deamidated β(2)M.
- An MS(3) strategy involving Collisionally Activated Dissociation (CAD) followed by ECD was developed and applied.
Main Results:
- Standard top-down ECD identified isoAsp at only one of three deamidation sites in β(2)M.
- The MS(3) CAD-ECD approach successfully identified isoAsp formation at all three deamidation sites.
- A linear correlation was observed between the abundance of the isoAsp diagnostic ion and the deamidation extent.
Conclusions:
- The MS(3) CAD-ECD approach significantly enhances the capability of top-down mass spectrometry for isoAsp detection in intact proteins.
- This method overcomes previous limitations in differentiating deamidation isomers at the intact protein level.
- The findings pave the way for improved quantitative analysis of protein deamidation and aging.
