A series of research that Identifies ADP-Ribosylation sites

It is well known that the ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs) and it is a crucial component in cell signaling, DNA repair, gene regulation and apoptosis. While researchers now can identify some ADP-ribose covalent acceptor sites, this work still shows some challenges.

ADP is easily lost during extraction or sample preparation for it is a post-translational modification. Another problem arises in identifying remaining ADP-riboslyated proteins, because the ADP-ribose modification is highly transient. Hydrolytic enzymes can degrade poly-ADP-ribose (PAR) to mono-ADP-ribose (MAR).

Rosenthal et al. explain that another stumbling block lies with a lack of suitable methods to identify and quantify the actual amino acid acceptor sites on ADP-ribosylated proteins. To meet the demand for better ADP-ribosylation detection, Rosenthal et al. optimized an approach to identify ADP-ribosylation sites using a hybrid ion trap-Orbitrap mass spectrometer. The researchers prepared ADP-ribosylated samples using the peptide biotin-KAARKSAPATGGVKKPHRYR (H3) and a mixture containing the four core histones as well as the H1 linker histone as full-length proteins.

The team chose an LTQ Orbitrap Velos ETD mass spectrometer (Thermo Scientific) coupled to a high-performance liquid chromatography (HPLC) system to finish their analysis. They used Proteome Discoverer software revision 1.4 (Thermo Scientific) to analyze data as well. The team combined higher-energy collisional dissociation (HCD) with electron-transfer dissociation (ETD). After the research, they came to the conclusion that this strategy produced more comprehensive coverage of ADP-ribosylation sites compared with HCD alone. In principle, putting ETD and HCD results together can increase cycle times, come out a lower number of identifications and assigned spectra, and is inefficient for complex samples. On the contrary, they used HCD after ETD on the same precursors only when one or more marker ions were present in the HCD spectra, or a product-dependent approach. Further analysis helped identify ADP-ribosylation sites with high reliability and confidence for in vitro modified proteins.

The researchers identified 167 ADP-ribosylated peptides and several ADP-ribose acceptor sites (Glu, Asp, Lys and Arg) on various different proteins. They think this research can help scientists understand the goal of reliably identifying ADP-ribose acceptor sites in complex samples.

More can be found here: http://www.cusabio.com/Recombinant-Protein/Recombinant-human-Medium-chain-specific-acyl-CoA-dehydrogenase-mitochondrial-11089628.html