Analyzing polyubiquitin chains upon ubiquitin activating enzyme inhibition from cell culture & tumor lysates using the Quanterix's single molecule array (Simoa) technology.

2013 Society for Laboratory Automation & Screening Annual Meeting, Orlando, FL

Ubiquitination is a post-translational modification process which involves the attachment of ubiquitin(Ub) to lysine residues on either substrate proteins or itself resulting in protein monoubiquitination or polyubiquitination. This modification controls cellular activity by either protein degradation of the polyubiquinated target protein via the proteasome or alterations of signaling processes. Ubiquitination is mediated by the sequential action of three enzymes: the E1 (ubiquitin-activating enzyme(UAE)), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Targeting this process has been our focus to develop an anti-cancer drug. Monitoring polyubiquitin expression as a biomarker or pharmacodynamic (PD) marker in cancer cell culture or xenograft models treated with inhibitors of UAE is, therefore, a useful tool to identify a potential therapeutic molecule.

We employed a new technology from Quanterix called Simoa (Single Molecule Array). This technology is capable of ultrasensitive protein measurements for monitoring the inhibition of polyubiquitination using femtoliter microwells to trap antibody captured molecules. Using both tissue culture and mouse xenograft samples and a complementary pair of commercially available antibodies against both linkage specific K48-polyUb and pan-polyUb, we first developed a protocol to establish a standard curve with recombinant K48-polyubiquitinated dihydrofolate reductase (polyUb5-DHFR). We then compared samples treated or untreated with UAE inhibitors from HCT116 and WSU-DLCL2 cell culture as well as the corresponding xenograft tumor lysates to measure changes in poly-K48Ub levels. Our results show that the Simoa technology is significantly more sensitive than other commercially available lysate-based platforms we have evaluated.