A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Shiori Kuraoka,Hideyuki Higashi,Yoshihiro Yanagihara,Abhijeet R Sonawane,Shin Mukai,Andrew K Mlynarchik,Mary C Whelan,Michael O Hottiger,Waqas Nasir,Bernard Delanghe,Masanori Aikawa,Sasha A Singh

doi:10.1016/j.mcpro.2021.100153

Shiori Kuraoka, Hideyuki Higashi + Show 10 more

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https://doi.org/10.1016/j.mcpro.2021.100153

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Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.

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ADP-ribosylation is a posttranslational modification (PTM) that is catalyzed by the polyadenosine diphosphate-ribose polymerase (PARP) enzyme family; referred to as the diphtheria toxin-like ADP-ribosyl transferases (ARTDs) [1]
NAD to proteins targets, whose amino acid acceptor sites have been identified as primarily aspartate, glutamate, lysine, arginine and serine; and, threonine, tyrosine, histidine and cysteine [2,3,4,5]
In the context IFN- -induced changes to mouse liver and spleen ADP-ribosylomes, we developed a set of novel ADPr annotation scores implemented as an enhancement to Proteome Discoverer 2.4

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ADP-ribosylation is a posttranslational modification (PTM) that is catalyzed by the polyadenosine diphosphate-ribose polymerase (PARP) enzyme family; referred to as the diphtheria toxin-like ADP-ribosyl transferases (ARTDs) [1]. ADP-ribosylation is a posttranslational modification (PTM) that is catalyzed by the polyadenosine diphosphate-ribose polymerase (PARP) enzyme family; referred to as the diphtheria toxin-like ADP-. The PARPs catalyze the transfer of the ADP-ribosyl (ADPr) moiety of NAD to proteins targets, whose amino acid acceptor sites have been identified as primarily aspartate, glutamate, lysine, arginine and serine; and, threonine, tyrosine, histidine and cysteine [2,3,4,5]. Proteins can be mono-ADP-ribosylated (MARylated) or poly-ADP-ribosylated (PARylated) [9], but only hydrolyzed forms of the PTM, for example the conversion of PAR to MAR peptides using poly-ADP-ribose glycohydrolase (PARG) [2, 10, 11] and the conversion of PAR/MAR to a phosphoribose using a phosphodiesterase [12], are conducive to mass spectrometry. The ADP-ribose is labile with collision induced dissociation (CID) and higher energy induced dissociation (HCD); producing lower mass fragments from the ADP-ribose moiety (m-ions) and the complementary peptide plus remaining ADP-ribose fragment (p-ions) [13]

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