Fragmentation behavior of Amadori-peptides obtained by non-enzymatic glycosylation of lysine residues with ADP-ribose in tandem mass spectrometry.

Mono- and poly-adenosine diphosphate (ADP)-ribosylation are common post-translational modifications incorporated by sequence-specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non-enzymatic ADP-ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori-compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix-assisted laser desorption/ionization (MALDI)-MS, the ADP-ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in-source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)-MS. The same fragmentation site also dominated the MALDI-PSD (post-source decay) and ESI-CID (collision-induced dissociation) mass spectra. The remaining phospho-ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b- and y-ion series. Cleavage of the ADP-ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor-ion scans (m/z 348.08), and thereby permits the identification of ADP-ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP-ribosyl group was stable, providing ADP-ribosylated c- and z-ions, and thus allowing reliable sequence analyses. Copyright 2010 John Wiley & Sons, Ltd.