Characterization of AMPylation on threonine, serine, and tyrosine using mass spectrometry

Yan Li, Rowaida Al-Eryani, Melanie L. Yarbrough, Kim Orth, Haydn L. Ball

Research output: Contribution to journalArticlepeer-review

23 Scopus citations


Recent studies have suggested that adenosine 5'-monophosphate (AMP) post-translational modification of proteins could represent a novel molecular signaling pathway. Mass spectrometric fragmentation characteristics of this modification have not previously been described and studied systematically. In this work, we therefore examined the fragmentation pattern of chemically synthesized peptides containing AMPylated Thr, Ser, and Tyr. The formation of characteristic ions and the influence of collision energy (CE) on the detection of characteristic ions and their relative peak intensity are reported. When peptide with AMPylated Ser/Thr underwent collision induced dissociation (CID), peaks at m/z 348.1, 136.1, and 250.1, fragments with AMP group attached, and fragments consistent with neutral loss of 347 Da were major characteristic ions; fragments consistent with neutral loss of 135 Da or 249 Da were weaker and not always detectable. The observations for Tyr AMPylation followed the same general patterns as those for Ser/Thr modification, with the exception that the ions detected for Tyr AMPylation did not include either the peak at m/z 348.1, or fragments with a mass shift of -347 Da. The results described in this paper highlight a series of diagnostic ions, which can be used not only to confidently identify the AMPylation site based on MS and MS/MS data, but also to selectively scan AMPylated peptides in complex protein mixtures.

Original languageEnglish (US)
Pages (from-to)752-761
Number of pages10
JournalJournal of the American Society for Mass Spectrometry
Issue number4
StatePublished - Apr 2011


  • AMPylation
  • Adenylation
  • Adenylylation
  • CID fragmentation
  • Post-translational modification

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy


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