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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
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A conventional procedure to reduce Asn deamidation artifacts during trypsin peptide mapping
Yekaterina Kori1, Rekha Patel1, Alyssa Neill1
1Product Characterization, Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, CT 06410, USA.
Summary
This study identifies Tris buffer as an optimal solution to minimize artifactual Asn deamidation during protein sample preparation. Using Tris buffer, especially at lower concentrations with acetonitrile, significantly reduces errors in quantifying protein deamidation levels.
Area of Science:
- Biochemistry
- Proteomics
- Analytical Chemistry
Background:
- Asparagine (Asn) deamidation is a critical post-translational modification impacting protein structure and function.
- Liquid chromatography-mass spectrometry (LC-MS) peptide mapping is standard for detecting Asn deamidation.
- Procedure-induced deamidation during sample preparation complicates accurate quantification.
Purpose of the Study:
- To evaluate common buffers for minimizing artifactual Asn deamidation during protein sample preparation.
- To optimize a procedure for accurate Asn deamidation analysis using LC-MS peptide mapping.
- To develop a universally applicable method for reducing sample preparation artifacts.
Main Methods:
- Evaluation of various buffers for their impact on Asn deamidation during sample preparation.
- Optimization of buffer concentration and addition of acetonitrile to reduce deamidation artifacts.
- Application of the optimized procedure for digesting a recombinant monoclonal antibody.
Main Results:
- Tris buffer significantly reduced artifactual Asn deamidation compared to other tested buffers.
- Lower concentrations of Tris buffer were particularly effective in minimizing deamidation.
- The addition of 10% acetonitrile further decreased deamidation artifacts.
- The optimized method successfully digested a monoclonal antibody with minimal induced deamidation.
Conclusions:
- Tris buffer, particularly at lower concentrations and with acetonitrile, provides an effective means to reduce procedure-induced Asn deamidation.
- This optimized protocol is practical for routine laboratory use, requiring no specialized reagents.
- The findings facilitate more accurate assessment of native Asn deamidation levels in proteins.
