Identifying ADP-Ribosylation Sites more accurately by various methods

Let's talk about something about ADP-ribosylation. It is catalyzed by ADP-ribosyltransferases (ARTs) and is quite an important element in apoptosis, DNA repair, cell signaling, gene regulation. Though researchers now can identify some ADP-ribose covalent acceptor sites, there are still some challenges. ADP is a post-translational modification and is easily lost during the state of extraction or sample preparation. Another problem happens in identifying remaining ADP-riboslyated proteins, because the ADP-ribose modification is very short. Hydrolytic enzymes can degrade poly-ADP-ribose to mono-ADP-ribose.

Researchers explain that another a tough nut is due to a lack of appropriate methods to identify and count the actual amino acid acceptor sites on ADP-ribosylated proteins. In order to optimize ADP-ribosylation detection, researchers use a hybrid ion trap-Orbitrap mass spectrometer to develope a method to identify ADP-ribosylation sites.

The researchers prepared some ADP-ribosylated samples and a mixture which is consist of the 4 core histones and the H1 linker histone as full-length proteins. In order to complete their analysis, the researchers chose an LTQ Orbitrap Velos ETD mass spectrometer coupled to a high-performance liquid chromatography system and used Proteome Discoverer software revision 1.4 to go through the data carefully.

The researchers took a strategy of combining higher-energy collisional dissociation with electron-transfer dissociation. The strategy produced more comprehensive coverage of ADP-ribosylation sites compared with only using HCD (higher-energy collisional dissociation). Instead of using the two methods together, the researchers used HCD followed by ETD on the same precursors only when one or more marker ions was present in the HCD spectra, or a product-dependent approach.

Further analysis showed more improved results using an HCD spectra ion trap product-dependent approach, or two-stage ion trap HCD-ETD. It helps researchers to identify ADP-ribosylation sites more accurately and be more confident in vitro modified proteins.

More can be found here: http://www.cusabio.com/ELISA-Kit/Human-Haptoglobin-Related-Protein-HPRELISA-Kit-1042050.html