Characterization of AMPylation on Threonine, Serine, and Tyrosine Using Mass Spectrometry

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.

Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach

“…Identification of AMPylation Sites-MS-based approaches for the identification of AMPylated peptides (22,23) take advantage of characteristic fragmentation patterns of the AMP moiety that upon collision-induced dissociation give rise to several well-established diagnostic ions. Although the methodology has the potential to scan AMPylated peptides in complex protein mixtures, this feature has not been further explored.…”

“…S4), and these were further validated through AMPylation with isolated HYPE E234G and ATP in place of Yn-6-ATP since the absence of a complex cellular lysate removes the need for enrichment. The MS/MS searches were then executed in analogous manner as for Az-RTB but taking advantage of published masses for native AMP adduct formation and its diagnostic ions (22,23). We observed a total of 12 autoAMPylation sites (Table S5), including the two sites previously assigned using Az-RTB methodology (Fig.…”

“…AMPylation profiling is not a trivial task (19), and several strategies have emerged over the past few years ranging from labeling with radioactive ATP (2, 3) and immunoprecipitation with AMPylation-specific antibodies (20,21) to mass spectrometry (MS) approaches focused on AMP fragmentation (22,23). Although these methods contributed significantly to developments in the field, they also suffer from certain drawbacks, including low sensitivity, high background, limited quantitative power, and limited amenability to high-throughput (HT) substrate identification.…”

Analysis of the Structure and Function of FOX-4 Cephamycinase

“…The matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis revealed two peaks, one at the expected mass and a prominent mass peak at the expected mass ϩ(329 Ϯ 8) Da (29), corresponding to the molecular weight of an adenylate (AMP) PTM (30,31). The same phenomenon was observed in three separate purifications of FOX-4 in different laboratories, all bearing different N-terminal tags that were proteolytically removed (His 6 , glutathione S-transferase, or His 9 -maltose binding protein).…”

A New Chemical Handle for Protein AMPylation at the Host–Pathogen Interface

“…[15] In turn, AMPylated substrate proteins have been identified by the combination of radioactive isotope labeling and targeted mass spectrometric analysis both on the MS and MS/MS level. [9][10][11]16] Two additional developments that complement the above-mentioned approaches include the elucidation of mass spectrometric fragmentation characteristics of AMPylation based on the analysis of synthetic peptides modified at serine, threonine, and tyrosine residues, [17] and the generation of a polyclonal antibody specific towards threonineAMPylation. [18] In a very recent report, a potentially more general, chemistry-based methodology for labeling AMP transferase substrates has been proposed by Hang, Orth and coworkers.…”