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

Proteomics. 2013 Mar;13(6):955-63. doi: 10.1002/pmic.201200344.

Abstract

Although the addition of a 5'-adenosine phosphodiester group to proteins, called adenylylation, has been known for decades, the possibility that adenylylation could be a molecular switch in cellular signaling pathways has emerged recently. The distinct mass shift upon adenylation of threonine or tyrosine residues renders it a good target for MS detection and identification; however, the fragmentation of adenylylated peptides derived from proteolytic digestion of adenylylated proteins has not yet been systematically investigated. Here, we demonstrate that adenylylated peptides show loss of parts of the adenosine monophosphate (AMP) upon different fragmentation techniques. As expected, causing the least fragmentation of the AMP group, electron transfer dissociation yields less complicated spectra. In contrast, CID and higher energy collision (HCD) fragmentation caused AMP to fragment, generating characteristic ions that could be utilized in the specific identification of adenylylated peptides. The characteristic ions and losses upon CID and higher energy collision fragmentation from the AMP group turned out to be highly dependent on which amino acid was adenylylated, with different reporter ions for adenylylated threonine and tyrosine. We also investigated how adenylylation is best incorporated into search engines, exemplified by Mascot and showed that it is possible to identify adenylylation by search engines.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Monophosphate / chemistry*
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Humans
  • Molecular Weight
  • Nucleotidyltransferases / chemistry
  • Peptide Fragments / chemistry*
  • Protein Processing, Post-Translational*
  • Proteomics / methods
  • Tandem Mass Spectrometry / methods*
  • cdc42 GTP-Binding Protein / chemistry
  • rab1 GTP-Binding Proteins / chemistry

Substances

  • Bacterial Proteins
  • Peptide Fragments
  • Adenosine Monophosphate
  • Nucleotidyltransferases
  • Rab1B protein, human
  • cdc42 GTP-Binding Protein
  • rab1 GTP-Binding Proteins