Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the Identification of ADP-Ribosylation Sites

ADP-ribosylation
of proteins alters their function or provides
a scaffold for the recruitment of other proteins, thereby regulating
several important cellular processes. Mono- or poly-ADP-ribosylation
is catalyzed by different ADP-ribosyltransferases (ARTs) that have
different subcellular localizations and modify different amino acid
acceptor sites. However, our knowledge of ADP-ribosylated proteins
and their acceptor amino acids is still limited due to the lack of
suitable mass spectrometry (MS) tools. Here, we describe an MS approach
for the detection of ADP-ribosylated peptides and identification of
the ADP-ribose acceptor sites, combining higher-energy collisional
dissociation (HCD) and electron-transfer dissociation (ETD) on an
LTQ-Orbitrap mass spectrometer. The presence of diagnostic ions of
ADP-ribose in the HCD spectra allowed us to detect putative ADP-ribosylated
peptides to target in a second LC–MS/MS analysis. The combination
of HCD with ETD fragmentation gave a more comprehensive coverage of
ADP-ribosylation sites than that with HCD alone. We successfully identified
different ADP-ribose acceptor sites on several in vitro modified proteins. The combination of optimized HCD and ETD methods
may be applied to complex samples, allowing comprehensive identification
of ADP-ribosylation acceptor sites.