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A mass spectrometry-based method to screen for α-amidated peptides
Zhenming An1, Yudan Chen, John M Koomen
1Department of Chemistry, University of South Florida, Tampa, FL 33620-5250, USA.
Proteomics
|November 23, 2011
Summary
Amidation, a key peptide modification, was identified using a novel precursor and α-amidated peptide pairing (PAPP) strategy. This method efficiently pairs peptides differing by 58 Da and similar retention times, aiding neuropeptide hormone discovery.
Area of Science:
- Biochemistry
- Proteomics
- Molecular Biology
Background:
- Amidation is a crucial post-translational modification at the C-terminus of approximately 50% of neuropeptide hormones.
- This modification, catalyzed by peptidylglycine α-amidating monooxygenase (PAM), involves the cleavage of a C-terminal glycine residue.
- α-amidated peptides and their precursors differ by 58 Da in mass but share similar RP-HPLC and MS/MS fragmentation patterns.
Purpose of the Study:
- To develop and implement a novel strategy for pairing α-amidated peptides with their glycine-extended precursors.
- To efficiently identify and characterize amidated neuropeptides in complex biological samples.
- To reduce the computational and manual effort required for peptide identification in mass spectrometry-based proteomics.
Main Methods:
- Cultured cells were treated with a PAM inhibitor to co-exist α-amidated peptides and their precursors.
- A precursor and α-amidated peptide pairing (PAPP) strategy was developed, utilizing LC-MS/MS data.
- Peptide pairs differing by 58 Da in mass and exhibiting similar RP-HPLC retention times were identified and validated by MS/MS fragmentation patterns.
Main Results:
- The PAPP method successfully identified α-amidated peptides from AtT-20 cell extracts.
- This approach significantly decreased the number of spectra requiring interpretation.
- Computational time for database searching was reduced, and manual interpretation of unidentified spectra was facilitated.
Conclusions:
- The PAPP strategy is an effective method for identifying α-amidated peptides and their precursors.
- This technique streamlines the analysis of post-translational modifications in proteomics.
- The findings contribute to a better understanding of neuropeptide hormone diversity and function.
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