Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

Uzoma, Ijeoma; Zhu, Heng

Readily generating a snapshot of global protein PTM profiles under various cellular conditions could be considered the Holy Grail for those researching PTMs. General PTM substrate identification strategies require enrichment from a cell extract sample followed by MS or in vitro assays using purified components. While both approaches have their strengths and weaknesses, a hybrid of the two is possible. The use of concentrated mammalian cell extracts in combination with protein microarrays can serve to identify PTM targets in a semiin vivo setting while alleviating the challenge of analyzing a complex mixture. Merbl and Kirschner generated cell extracts that replicate the mitotic checkpoint and anaphase release to identify differentially regulated polyubiquitylation substrates [21] . The synchronized cell extracts were incubated with Invitrogen's Human ProtoArray composed of 8000 proteins and the resulting polyubiquitylated proteins were detected with antibodies directed to ubiquitin chains [21] . The authors expected to recover substrates of the anaphase promoting complex (APC), the major ubiquitin ligase in mitosis and G1. To differentiate polyubiquitylation substrates of the APC from other ligases, Merbl and Kirschner designed three experimental set ups. All cell extracts were arrested with nocodozole as the control which inhibits the APC, in the second condition the sample was released from checkpoint arrest with the addition of UbcH10, an E2 ligase, and the final condition was supplemented with both UbcH10 and a specific inhibitor of APC. Approximately 132 proteins were differentially polyubiquitylated, 11 of which were known APC substrates, confirming the validity on the experimental design. Validation studies performed in rabbit reticulocyte lysate confirmed the degradation/ ubiquitylation of 7 novel APC substrates [21] . This study demonstrates the efficacy of using protein microarrays in combination with cell extracts to recapitulate the global PTM signature in a specific cellular state.