Proteomics

Our lab has developed multiple non-biased proteomics approaches to identify ADP-ribosylation sites of different classes at the proteome level, revealing amino acid specificity of viral macrodomain.

Daniels CM, Ong SE, Leung AK. Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells. J Proteome Res. 2014 Aug 1;13(8):3510-22. [Abstract/PDF]

McPherson RL, Ong SE, Leung AKL. Ion-Pairing with Triethylammonium Acetate Improves Solid-Phase Extraction of ADP-Ribosylated Peptides. J Proteome Res. 2020 Feb 7;19(2):984-990. [Abstract/PDF]

Informatics

We developed a widely accessed database of ADP-ribosylated proteins and sites (ADPriboDB) with over 700,000 hits since its first release in April 2016.

Vivelo CA, Wat R, Agrawal C, Tee HY, Leung AK. ADPriboDB: The database of ADP-ribosylated proteins. Nucleic Acids Res. 2016 Jan 4;45(D1):D204-D209. [Abstract/PDF]

Ayyappan V, Wat R, Barber C, Vivelo CA, Gauch K, Visanpattanasin P, Cook G, Sazeides C, Leung AKL. ADPriboDB 2.0: an updated database of ADP-ribosylated proteins. Nucleic Acids Res. 2021 Jan 8;49(D1):D261-D265. [Abstract/PDF]

End-Labeling tools ELTA and beyond

Our current understanding of DNA and RNA has been advanced by simple bioconjugation technologies that add tiny beacons or chemical handles. Without these technologies, it would have been impossible to have the sequencing ability to characterize the human genome and its functions.

Although PAR is chemically similar to DNA and RNA, a simple bioconjugation technology did not exist for PAR. The lack of this technology for PAR makes it difficult to take advantage of the wide repertoire of molecular biology tools already available to measure, detect and enrich for DNA or RNA.

We recently developed the first method called ELTA to enzymatically label 2’ terminus of many forms of ADP-ribose. The technique opens up the possibility for profiling the length of ADP-ribosylation, measurement of the protein-PAR affinity and enrichment of endogenous ADP-ribosylated substrates for site identification at sub-femtomole sensitivity.

In addition, we developed a chemical technique to label 1" terminus of poly(ADP-ribose) to complement ELTA. With the ability to label both polymer ends, we now have the necessary building blocks for synthetic biology of PAR.

Ando Y, Elkayam E, McPherson RL, Dasovich M, Cheng SJ, Voorneveld J, Filippov DV, Ong SE, Joshua-Tor L, Leung AKL. ELTA: Enzymatic Labeling of Terminal ADP-Ribose. Mol Cell. 2019 Feb 21;73(4):845-856.e5. [Abstract/PDF]

Abraham R, McPherson RL, Dasovich M, Badiee M, Leung AKL*, Griffin DE*. Both ADP-Ribosyl-Binding and Hydrolase Activities of the Alphavirus nsP3 Macrodomain Affect Neurovirulence in Mice. mBio. 2020 Feb 11;11(1):e03253-19. (*co-corresponding) [Abstract/PDF]

Chemical Probe PARprolink

Using ELTA, we developed the chemical probe to identify direct PAR binders, enabling us to conduct the first census of human PAR-binding proteome. Our census revealed that these PAR binders are enriched in various biomolecular condensates (e.g., stress granules, nucleoli and DNA repair foci).

Using specific probes, we identified dozens of proteins that prefer binding to long over short PAR length. These data further support the PAR code hypothesis, where poly(ADP-ribose) directs condensate formation in cells.

Dasovich M, Beckett MQ, Bailey S, Ong SE, Greenberg MM, Leung AKL. Identifying Poly(ADP-ribose)-Binding Proteins with Photoaffinity-Based Proteomics. J Am Chem Soc. 2021 Mar 3;143(8):3037-3042. [Abstract/PDF]