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J Am Soc Mass Spectrom. 2011 Apr;22(4):752-61. doi: 10.1007/s13361-011-0084-1. Epub 2011 Feb 24.

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

Author information

1
Protein Chemistry Technology Center, Department of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas, TX, USA.

Abstract

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.

PMID:
21472612
PMCID:
PMC3391576
DOI:
10.1007/s13361-011-0084-1
[Indexed for MEDLINE]
Free PMC Article

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