Design of antibody microarrays for global profiling of membrane proteins and soluble proteins

Abstract: Antibody-based microarrays have emerged as an established proteomic technology allowing multiplexed and sensitive profiling of complex proteomes, in a high-throughput and miniaturized manner. Recently, numerous applicative efforts have been pursued generating disease-associated protein signatures that now could be further explored for improved disease diagnostics, prognostics and classification. The aim of this thesis, based on five original papers, was to develop the antibody microarray methodology even further, to allow for global proteome analysis. Accordingly, a miniaturization of the array features was established, allowing for high-density arrays to be fabricated. We showed that sensitive detection of protein analytes in pure system as well as complex serum samples could be achieved using this miniaturized recombinant antibody nanoarray set-up. In additional globalization efforts, we demonstrated that two, novel, recombinant antibody microarray set-ups, based on human scFv antibody fragments, could be designed for membrane protein profiling of intact cells and cell/tissue extracts, respectively. This will provide us with unique and novel means to delineate the membrane proteome, previously proven to be to be difficult to address using conventional proteomic approaches. Taking advantage of these technological developments in a follow-up applicative study, differentially expressed membrane proteins and water-soluble proteins were readily identified in preeclamptic placenta vs. normal placenta. The data showed that candidate disease-associated tissue protein signatures could be identified, that could help to decipher the complex features of preeclampsia at the molecular level. Finally, we demonstrated how a wide variety of protein analytes could be targeted in mantle cell lymphoma, adopting genome-based affinity proteomics, using a novel reverse-phase antibody microarray set-up. Altogether, these technological improvements will allow us to gain further insights into complex molecular pathways in health and disease.