Adenylylation, MS, and proteomics--Introducing a "new" modification to bottom-up proteomics

Area of Science:

• Biochemistry
• Proteomics
• Molecular Signaling

Background:

• Adenylylation, the addition of a 5'-adenosine phosphodiester group to proteins, is a known modification.
• Recent research suggests adenylylation acts as a molecular switch in cellular signaling.
• Mass spectrometry (MS) is suitable for adenylylation detection due to mass shifts, but peptide fragmentation patterns are understudied.

Purpose of the Study:

• To systematically investigate the fragmentation of adenylylated peptides.
• To identify characteristic ions and losses for specific adenylylation site identification.
• To optimize adenylylation incorporation into protein identification search engines.

Main Methods:

• Proteolytic digestion of adenylylated proteins.
• Analysis of adenylylated peptides using various MS fragmentation techniques: Electron Transfer Dissociation (ETD), Collision-Induced Dissociation (CID), and Higher-Energy Collisional Dissociation (HCD).
• Incorporation of adenylylation into Mascot search engine parameters.

Main Results:

• Adenylylated peptides exhibit characteristic losses of adenosine monophosphate (AMP) fragments during fragmentation.
• ETD fragmentation results in less complex spectra with minimal AMP fragmentation.
• CID and HCD fragmentation produce specific reporter ions dependent on whether threonine or tyrosine residues are adenylylated, aiding identification.
• Adenylylation can be successfully incorporated and identified using standard search engines like Mascot.

Conclusions:

• Fragmentation patterns of adenylylated peptides provide specific identifiers for adenylylation.
• CID and HCD fragmentation are valuable for identifying adenylylated threonine and tyrosine residues.
• Computational tools like Mascot can effectively identify adenylylation in proteomic datasets.