Global Analysis of Protein Activities Using Proteome Chips

Abstract

To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

Diels–Alder Ligation of Peptides and Proteins

“…[64] Because the immobilized protein can adopt a variety of unpredictable orientations upon binding to the surface, these methods may lead to insufficient exposure of functional domains of a particular protein, rendering weak signals in further interactions with other analytes. The incorporation of recombinant affinity tags into specific sites of the protein molecule addresses the orientation issue (for instance, recombinant His-tagged proteins that binds to Ni-NTAcoated slides [68] ). However the interactions of the tags, like the other approaches of noncovalent immobilization, are often reversible and may not be stable over the course of subsequent assays, resulting in graduate depletion of the protein from the microarray surface.…”

A pooling-deconvolution strategy for biological network elucidation

“…We tested PI-Deconvolution using yeast proteome microarrays 11,12 , which contain 4,088 purified Saccharomyces cerevisiae proteins (as glutathione S-transferase fusions) immobilized on nitrocellulose-coated glass slides. For this purpose, 15 (~16=2 4 ) V5 epitope-tagged bait proteins were employed (Fig.…”

Next generation microfluidic platforms for high-throughput protein biochemistry

“…Protein arrays were predicted to impact our understanding of protein structure and function, in the same way that DNA arrays provided insight into gene expression and regulation [4,5]. Unfortunately generating and applying protein arrays had one significant impediment: proteins!…”