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Published on: December 12, 2013
Detecting D-amino acid-containing neuropeptides using selective enzymatic digestion
Michael A Ewing1, Jane Wang, Sarah A Sheeley
1Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801, USA.
Analytical Chemistry
|March 18, 2008
Summary
Mass spectrometry struggles to detect D-amino acid post-translational modifications (PTMs). A new method uses an enzyme to selectively degrade non-D-amino acid peptides, enabling the identification of rare D-amino acid-containing peptides (DAACPs).
Area of Science:
- Biochemistry
- Proteomics
- Neuroscience
Background:
- Neuropeptides are crucial signaling molecules.
- Post-translational modifications (PTMs) significantly alter neuropeptide function.
- D-amino acid formation is a PTM challenging to detect with mass spectrometry (MS) due to lack of mass shift.
Purpose of the Study:
- To develop a novel method for identifying D-amino acid-containing peptides (DAACPs) in complex biological mixtures.
- To overcome the limitations of MS in detecting D-amino acid PTMs.
- To enable the discovery of previously uncharacterized DAACPs.
Main Methods:
- Enzymatic digestion using microsomal alanyl aminopeptidase.
- Selective degradation of peptides lacking D-amino acids at the N-terminal second position.
- Comparison of mass spectrometry data before and after enzymatic treatment.
Main Results:
- The enzymatic digestion method successfully identified DAACPs.
- This approach allows for the detection of DAACPs even when present in low quantities within complex peptide mixtures.
- A known DAACP from Aplysia californica abdominal ganglion was identified, validating the protocol.
Conclusions:
- Microsomal alanyl aminopeptidase serves as a valuable discovery-enabling agent for DAACP identification.
- This method enhances the characterization of neuropeptide PTMs.
- The protocol offers a powerful tool for advancing proteomics and neuroscience research.
